Small molecular inhibitors of RAD51 recombinase and methods thereof

ABSTRACT

The invention includes compositions comprising a selective small-molecule inhibitor of RAD51 recombinase and a pharmaceutically acceptable carrier. The invention further includes methods of treating or preventing cancer in a subject, comprising the step of administering to the subject the compositions contemplated within the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §371 national phase applicationof, and claims priority to, International Application No.PCT/US12/25267, filed Feb. 15, 2012, and published under PCT Article21(2) in English, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 61/447,410, filed on Feb. 28, 2011, all ofwhich applications are hereby incorporated by reference in theirentireties herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersCA100839 and MH084119 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

As a high-fidelity recombination that is evolutionarily conserved frombacteria to mammals, homologous recombination plays an essential role inmaintaining genome integrity. Homologous recombination plays a criticalrole in the repair of DNA double-strand breaks and interstrandcrosslinks, the most harmful types of DNA lesions (Helleday et al.,2007, “DNA double-strand break repair: Prom mechanistic understanding tocancer treatment”, DNA Repair (Amst); Krogh & Symington, 2004, Annu.Rev. Genet, 38:233-71; San Filippo et al., 2008, Ann. Rev. Biochem,77:229-57). Mutations in homologous recombination genes may cause cancerand genetic abnormalities related syndromes, such as Down's, Werner'sand Klinefelter's syndromes (Hoeijmakers, 2001, Nature 411:366-74;Helleday el al, 2007, DNA Repair 6:923-35).

RAD51 recombinase (human sequence, SEQ ID NO:1), an ortholog of E. coliRecA, is a key protein in homologous recombination in mammalian cells,RAD51 promotes the repair of double-strand breaks, the most harmful typeof DNA lesion. Double-strand breaks are induced by various chemicalagents and ionizing radiation, and are also formed during the repair ofinterstrand crosslinks, Once double-strand breaks are formed, (hey areprocessed first by exonucleases to generate extensive ssDNA tails (Cejkaet al., 2010, Nature 467:112-16; Mimitou & Symington, 2009, “DNA endresection: many nucleases make light work”, DNA Repair (Amst) 8:983-95),Then RAD51 protein binds these ssDNA tails forming helical nucleoproteinfilaments that promote in search for homologous dsDNA sequences(Kowalczykowski, 2008, Nature 453:463-66), Once homologous dsDNAsequences are found, RAD51 promotes DNA strand exchange between thessDNA that resides within the filament and homologous dsDNA, i.e., aninvasion of ssDNA into homologous DNA duplex that results in thedisplacement of the identical ssDNA from the duplex and formation of ajoint molecule. Joint molecules, key intermediates of DSB repair,provide both the template and the primer for DNA repair synthesis thatis required for double-strand break repair (Paques & Haber, 1999,Microbiol. Mol. Biol. Rev. 63:349-404).

By promoting DNA strand exchange, RAD51 plays a key role in homologousrecombination. The protein is evolutionarily conserved frombacteriophages to mammals. In all organisms RAD51 orthologs play animportant role in DNA repair and homologous recombination (Krough &Symington, 2004, Annu. Rev. Genet. 38:233-71; Helleday et al., 2007, DNARepair 6:923-35; Huang et al., 1996, Proc. Natl. Acad. Sci. USA93:4827-32; Tsuzuki et al., 1996, Proc. Natl. Acad. Sci. USA 93,6236-40). However, only in higher eukaryotes does Rad51 become essentialfor cell viability. The knockout of the murine RAD51 gene causedembryonic lethality of homozygoes (San Filippo et al., 2008, Ann. Rev.Biochem. 77:229-57). Murine embryonic fibroblasts became prematurelysenescent in tissue culture and did not proliferate for more than a fewgenerations. Rad51 inactivation is detrimental for proliferation of thechicken DT-40 cells, as well (Li et al., 2009, Biochemistry 48:6805-10).

RAD51 was found to be overexpressed in many tumors, including familialBRCAI-deficient breast tumors (Raderschall et al., 2002, Cancer Res62:219-25; Xia et al., 1997, Mol. Cell. Biol. 17:7151-58; Maacke et al.,2000, Intl. J. Cancer 88:907-13). Overexpression of RAD51 is thought torescue homologous recombination by compensating for the lack offunctional BRCAI or other DNA repair proteins. Because RAD51overexpression may contribute to chemoresistance and radioresistance ofhuman cancers (Ito et al., 2005, J. Gene Med. 7:1044-52), this proteinrepresents an important target for anti-cancer therapy. Identificationand use of RAD51 inhibitors may lead to development of novel combinationanticancer therapies. Since homologous recombination plays an importantrole in the repair of double-strand breaks and interstrand crosslinks,efficiency of traditional anticancer therapies, which widely useionizing radiation and other double-strand-breaking andintrastrand-crosslinking agents, may be increased by inhibitinghomologous recombination in cancer cells by virtue of inhibiting theaction of RAD51. Furthermore, inhibitors that block specific activitiesof RAD51, like DNA strand exchange or ATP hydrolysis, may be useful inthe investigation of the cellular functions of this protein. Recently,small molecules inhibitors were employed in several studies toinvestigate the activity of RAD51 in homologous recombination (Li etal., 2009, Biochemistry 48:6805-10; Ishida et al., 2009, Nucl. AcidsRes. 37:3367-76). However, so far no specific inhibitors of RAD51 havebeen disclosed in the art.

There is a need in the art to identify novel small molecule inhibitorsof human RAD51 recombinase. The present invention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of Formula (1):

wherein R¹ and R² are independently selected from the group consistingof H, C₁-C₆ alkyl, C₁-C₆ substituted alkyl, phenyl, substituted phenyl,heteroaryl, substituted heteroaryl, heterocyclyl, substitutedheterocyclyl, —(C₁-C₆)alkylene-phenyl, —(C₁-C₆) alkylene-substitutedphenyl, —(C₁-C₆)alkylene-heteroaryl, and —(C₁-C₆)alkylene-substitutedheteroaryl; R³ is H, C₁-C₆ alkyl, O(C₁-C₆ alkyl), F, Cl, Br or I; or asalt thereof.

In one embodiment, R¹ is phenyl, substituted phenyl, heteroaryl, orsubstituted heteroaryl. In another embodiment, R¹ is selected from thegroup consisting of phenyl, o-tolyl, m-tolyl, p-tolyl, o-methoxyphenyl,m-methoxyphenyl, p-methoxyphenyl, o-halophenyl, m-halophenyl,p-halophenyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl, 2-pyridinyl,3-pyridinyl, and 4-pyridinyl.

In one embodiment, R² is C₁-C₆ alkyl, phenyl, substituted phenyl,heteroaryl, substituted heteroaryl, —(C₁-C₆)alkylene-phenyl,—(C₁-C₆)alkylene-substituted phenyl, —(C₁-C₆)alkylene-heteroaryl, or—(C₁-C₆)alkylene-substituted heteroaryl. In another embodiment, R² isC₁-C₆ alkyl, phenyl, substituted phenyl, heteroaryl, substitutedheteroaryl, —(C₁-C₆)alkylene-phenyl, or —(C₁-C₆)alkylene-substitutedphenyl. In yet another embodiment, R² is methyl, ethyl, n-propyl,isopropyl, phenyl, o-tolyl, m-tolyl, p-tolyl, benzyl or substitutedbenzyl.

In one embodiment, R³ is H, C₁-C₆ alkyl, —O(C₁-C₆ alkyl), F or Cl. Inanother embodiment, R³ is H, methyl, ethyl, methoxy or ethoxy. In yetanother embodiment, R³ is H.

In one embodiment, the compound is selected from the group consistingof:

-   (E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1a)

-   (E)-3-ethyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1b)

-   (E)-2-(2-(pyridin-3-yl)vinyl)-3-(m-tolyl)quinazolin-4(3H)-one (1c)

mixtures thereof and salts thereof.

In one embodiment, the composition further comprises a chemotherpeuticagent. In another embodiment, the agent is selected from the groupconsisting of an alkylating agent, antimetabolite, anthracycline, plantalkaloid, plant terpenoid, topoisomerase inhibitor, antineoplasticagent, and combinations thereof.

The invention also includes a method of treating or preventing cancer ina subject in need thereof. The method comprises administering to thesubject a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a pharmaceutically effective amount of a compoundselecting from the group consisting of Formula (1), Formula (2), Formula(3), a salt thereof, and combinations thereof:

wherein in (1): R¹ and R² are independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ substituted alkyl, phenyl,substituted phenyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, —(C₁-C₆)alkylene-phenyl,—(C₁-C₆)alkylene-substituted phenyl, —(C₁-C₆)alkylene-heteroaryl, and—(C₁-C₆)alkylene-substituted heteroaryl; R³ is H, C₁-C₆ alkyl, O(C₁-C₆alkyl), F, Cl, Br or I; or a salt therof. The method further comprisesadministering to the subject a treatment selected from the groupconsisting (i) radiation therapy, and (ii) a pharmaceutical compositioncomprising a pharmaceutically effective amount of a chemotherapeuticagent; whereby treating or preventing the cancer in the subject.

In one embodiment, the compound is selected from the group consisting of(E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1a),(E)-3-ethyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1b),(E)-2-(2-(pyridin-3-yl)vinyl)-3-(m-tolyl)quinazolin-4(3H)-one (1c),1,4,10-trihydroxy-5-(hydroxymethyl)-8-methyl-3,7-dioxo-3,7-dihydro-1H-benzo[6,7][1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde(2),1,4-dihydroxy-10-methoxy-5,8-dimethyl-3,7-dioxo-3,7-dihydro-1H-benzo[6,7][1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde(3), a salt thereof, and mixtures thereof.

In one embodiment, the administering to the subject of the compound isperformed at least 24 hours prior to the administering to the subjectthe radiation therapy or the chemotherapeutic agent. In anotherembodiment, the administering to the subject of the compound isperformed at least 12 hours prior to the administering to the subjectthe radiation therapy or the chemotherapeutic agent. In yet anotherembodiment, the administering to the subject the radiation therapy orthe chemotherapeutic agent. In yet another embodiment, the administeringto the subject of the compound is performed at least 3 hours prior tothe administering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, the administering tothe subject of the compound is performed at least 1 hour prior toadministering to the subject the radiation therapy or thechemotherapeutic agent.

In one embodiment, the composition comprising the compound isco-administered to the subject with the radiation therapy or thecomposition comprising the chemotherapeutic agent. In anotherembodiment, the compound and the chemotherapeutic agent areco-formulated in a pharmaceutical composition. In yet anotherembodiment, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIGS. 1A-1B, illustrates the process of measuringRAD51-promoted DNA strand exchange using the FRET-based assay. FIG. 1Aillustrates the reaction scheme. The terms “FLU” and “BHQ1” denotefluorescein and black hole quencher 1, respectively. Broken-line andsolid-line arrows denote fluorescein emission at 521 nm before and afterDNA strand exchange, respectively. The excitation wavelength was 490 nm.FIG. 1B is a graph that illustrates the kinetics of DNA strand exchangepromoted by RAD51. The fluorescence intensity was expressed in arbitraryunits (AU). “Homologous DNA” and “Heterologous DNA” denote reactionswith homologous (SEQ ID NO: 2, 48-mer) and heterologous ssDNA (SEQ IDNO:5, 48-mer), respectively.

FIG. 2, comprising FIGS. 2A-2B, illustrates selected RAD51 inhibitorsidentified by HTS.

FIG. 3, comprising FIGS. 3A-3E, illustrates the secondary screening ofthe RAD51 inhibitors using the D-loop assay. FIG. 3A is a schemeillustrating the D-loop formation promoted by RAD51 . The asteriskdenotes the ³²P label. FIG. 3B is a reproduction of an electrophoresisgel that illustrates the analysis of 17 compounds selected by HTS. RAD51(1 μM) was incubated with a 90-mer ssDNA (3 μM) (SEQ ID NO:6) to formthe filament followed by addition of indicated compounds (100 μM). Jointmolecule (D-loop) formation was initiated by addition of pUC19supercoiled dsDNA (50 μM). The DNA products were analyzed byelectrophoresis in a 1% agatose gel. The control was carried out underidentical conditions except that no tested compounds on the yield ofjoint molecules. The extent of D-loop formation in the absence ofinhibitors, 40.3%, was expressed as 100% of D-loop formation efficiency.Experiments were repeated at least three times: error bars representstandard deviation (standard deviation). FIG. 3D comprises reproductionsof electrophoresis gels that illustrate the effect of Compound B02concentration on the DNA strand exchange activity of RAD51 and RecA.After incubation of RAD51 (0.3 μM) or RecA (0.3 μM) with Compound B02 inindicated concentrations for 30 min, 0.9 μM ssDNA (SEQ ID NO:6, 90 mer)was added to form RAD51 nucleoprotein filament for 15 min. The D-loopformation was initiated by addition supercoiled pUC19 dsDNA (15 μM). Thecontrol reactions containing no proteins are shown in lane 1 and 10. InFIG. 3E, the yield of joint molecules (D-loops) was plotted as a graph.The extents of D-loop formation in the absence of Compound B02, 32% and33.7% for RAD51 and RecA, respectively, were expressed as 100% of D-loopformation efficiency. Experiments were repeated at least three times;error bars represent S.E.M. (standard error of the mean).

FIG. 4 is a graph illustrating the IC₅₀ of RAD51 inhibition by fourselected compounds determined in the D-loop assay. RAD51 (1 μM) wasincubated with a 90-mer ssDNA (3 μM) (SEQ ID NO:6) to form the filamentfollowed by addition of Compounds A03, A04, A10, and B02 in indicatedconcentrations. After a 30-min incubation, D-loop formation wasinitiated by addition of pUC19 supercoiled dsDNA (50 μM). The DNAproducts were analyzed by electrophoresis in a 1% agarose gel.Experiments were repeated at least three times; error bars representstandard deviation.

FIG. 5, comprising FIGS. 5A-5D, is a series of graphs illustrating thespecificity of RAD51 inhibition by Compounds A03, A04, A10, and Bo2.RAD51 (1 μM) or RecA (1 μM) was incubated with a 90-mer ssDNA (3 μM)(SEQ ID NO:6) for 15 min (for RAD51) or 5 min (for RecA) to form thenucleoprotein filament. Then, tested compounds in indicatedconcentrations were added and incubation continued for 30 min. TheD-loop formation was initiated by addition of pUC19 supercoiled dsDNA(50 μM) and continued for 15 min (for RAD51) or 3 min (for RecA). TheDNA products were analyzed by electrophoresis in a 1% agarose gel. Theyield of joint molecules (D-loops) was plotted as a graph. Experimentswere repeated at least three times; error bars represent standarddeviation.

FIG. 6, comprising FIGS. 6A-6C, illustrates the effect of Compounds A03,A04, A10 and B02 on branch migration activity of RAD54. FIG. 6A is ascheme that illustrates a scheme of branch migration promoted by RAD54.The asterisk denotes the ³²P label. FIGS. 6B and 6C: branch migrationwas initiated by adding RAD54 (100 nM) to the mixtures containingPX-junctions (33 nM, molecules) and the small molecule inhibitors (inindicated concentrations). DNA products were analyzed by electrophoresisin 8% polyacrylamide gels. For each inhibitor concentration, the extentof branch migration was determined after 5 min of reaction (linearphase). The results are presented as graphs in FIGS. 6B and 6C.Experiments were repeated at least three times; error bars representstandard deviation.

FIG. 7, comprising FIGS. 7A-7C, illustrates the analysis ofStructure-Activity Relationship (SAR) of Compound B02. FIG. 7Aillustrates the structures of Compound B02 and its derivatives. FIG. 7Bis a reproduction of an electrophoresis gel that illustrates the effectof indicated B02 derivatives on D-loop formation by RAD51, RAD51 (1 μM)was incubated with a 90-mer ssDNA (3 μM) (SEQ ID NO:6) for 15 minfollowed by addition of indicated compounds (50 μM). After a 30-minincubation, the D-loop formation was initiated by addition of 50 μMsupercoiled pUC19 dsDNA. The DNA products were analyzed byelectrophoresis in a 1% agarose gel. The control reaction was performedunder the identical conditions, except that no tested compounds wereadded. FIG. 7C is a graph that illustrates the yield of joint molecules(D-loops). Experiments were repeated at least three times; error barsrepresent standard deviation.

FIG. 8, comprising FIGS. 8A-8C, illustrates the effect of Compounds C3aand C3b on DNA strand exchange activity of RAD51 and RecA. FIG. 8A is areproduction of an electrophoresis gel illustrating the effect of C3a onthe DNA strand exchange activity of RAD51 and RecA. The nucleoproteinfilaments were formed by incubating RAD51 (1 μM) or RecA (1 μM) withssDNA (3 μM), then C3a was added in indicated concentrations andincubation continued for 30 min. D-loop formation was initiated byadding pUC19 supercoiled dsDNA (50 μM). The DNA products were analyzedby electrophoresis in a 1% agarose gel. The data from FIG. 8A wasplotted as a graph in FIG. 8B. The yield of D-loop formation in theabsence of C3a was expressed as 100%; the actual yield was 45.1% and34.2%, for RecA and RAD51, respectively, FIG. 8C illustrates the effectof C3b on the DNA strand exchange activity of RAD51 and RecA. Thereactions were carried out as in FIG. 8A; the data are plotted as agraph. The yield of D-loop formation in the absence of C3b was expressedas 100%; the actual yield was 48.1% and 31.6%, for RecA and RAD51,respectively. Experiments were repeated at least three times; error barsrepresent standard deviation.

FIG. 9 is an illustration of the structure of(E)-3-benzyl-2-(2-(pyridin-3-yl) vinyl)quinazolin-4(3H)-one) (EBVQ orCompound B02).

FIG. 10, comprising FIGS. 10A-10C, illustrates the inhibition byCompound B02 of the three strand exchange promoted by RAD51 protein.FIG. 10A is a schematic representation of DNA strand exchange betweenφX174 circular ssDNA and linear φX174 dsDNA (linearized by ApaL1restriction endonuclease). FIG. 10B is a reproduction of anelectrophoresis gel illustrating the effect of the Compound B02concentration on the efficiency of three strand exchange assay promotedby RAD51, RAD51 was incubated with indicated concentration of CompoundB02 (lane 2-9) for 30 min; then φX174 circular ssDNA and RPA were addedin turn, each addition was followed by a 5-min incubation; the strandexchange was initiated by addition of linear φX174 dsDNA. The DNAproducts were analyzed by electrophoresis in a 1% agarose gel. FIG. 10Cis a graph illustrating data from FIG. 10B. Experiments were repeated atleast three times; error bars represent standard error of the mean.

FIG. 11, comprising FIGS. 11A-11C, illustrates the finding that theorder of Compound B02 addition affects the efficiency of D-loopformation promoted by RAD51, FIG. 11A is a reproduction of anelectrophoresis gel illustrating the effect of the order of addition ofCompound B02. The numbers above the arrows indicate time of incubation.(I) Compound B02 (20 μM) was added after the RAD51-ssDNA filamentformation; (II) Compound B02 (20 μM) was added RAD51 before addition ofssDNA. FIG. 11B is a graph illustrating the analysis of joint moleculesby electrophoresis in a 1% agarose gel. FIG. 11C is a graph in which therelative inhibition of joint molecule formation by Compound B02 isexpressed as the ration of the joint molecules formed by RAD51 afterCompound B02 treatment to those formed by RAD51 without Compound B02treatment. “l” denotes the time period between the addition of CompoundB02 and dsDNA. Experiments were repeated at least three times; errorbars represent standard error of the mean.

FIG. 12, comprising FIG. 12A-12B, illustrates the finding that CompoundB02 inhibits ssDNA binding of RAD51. FIG. 12A is a reproduction of anelectrophoresis gel in which RAD51 (1 μM) was incubated with ³²P-labeledssDNA (SEQ ID NO:6, 90 mer) (2.5 μM, nt) in buffer containing indicatedNaCl concentration either in the absence (lanes 2-7) or presence (lanes8-13) of Compound B02 (25 μM). RAD51-ssDNA complexes were analyzed byelectrophoresis in a 10% polyacrylamide gel. The results in FIG. 12Awere shown as a graph in FIG. 12B. Lane 1 show migration of free ssDNA.Experiments were repeated at least three times; error bars representstandard error of the mean.

FIG. 13 is a graph illustrating inhibition of the DNA-dependent ATPaseactivity of RAD51 by Compound B02 in a concentration-dependent manner.RAD51 (1 μM) was incubated with Compound B02 in indicated concentrationfor 30 min in buffer containing 25 mM Tris-acetate (pH 7.5), 1 mM DTT,100 μg/ml bovine serum albumin (BSA), 2 mM Mg(OAc)₂, 0.1 mM ATP and 10μCi[γ-³²P]ATP(6000 Ci/mmole), then ssDNA (3 μM) (SEQ ID NO:6, 90 mer)was added to initiate ATP hydrolysis. After 1.5 h incubation, thesamples were analyzed by PEI-TLC. Experiments were repeated at leastthree times; error bars represent standard error of the mean.

FIG. 14, comprising FIGS. 14A-14B, illustrate the inhibition by CompoundB02 of the coaggregation of dsDNA with the RAD51-ssDNA filament. FIG.14A is a scheme illustrating dsDNA coaggregation. FIG. 14B is a graphillustrating inhibition of the coaggregation of dsDNA and RAD51-ssDNAfilament by Compound B02. To form the RAD51-ssDNA filaments, RAD51 (1μM) and ssDNA (SEQ ID NO:7, 94 mer) (3 μM), nt) were incubated for 15min. After filament formation, NaCl was added in indicatedconcentrations and coaggregation was initiated immediately by additionof ³²P-labeled linear pUC19 dsDNA (linearized by BamHI restrictionendonuclease) (25 μM, nt). Experiments were represented at least threetimes; error bars represent standard error of the mean.

FIG. 15, comprising FIGS. 15A-15C, illustrates the finding that CompoundB02 inhibits. DSB-induced homologous recombination in human cells. FIG.15A is a scheme illustrating the process of measuring the frequency ofhomologous recombination in human cells using the DRGFP reporter system.FIG. 15B is a set of panels (panels 1-6) illustrating the effect ofCompound B02 on the repair of I-SceI-induced DSBs in 293 HEK cellscarrying the chromosomally located DR-GFP reporter, as determined usingflow cytometry. Green fluorescence (GRN-Hlog, indicated as “G” in thefigure) was plotted against red fluorescence (RED-Hlog, indicated as “R”in the figure) for the sample of 10,000 cells. The GTP-positivepopulation is denoted by the elliptical MI marker. Cells withI-SceI-induced DSBs were either untreated (panel 2) or treated withCompound B02 5 μM (panel 3), 10 μM (panel 4), or 20 μM (panel 5). As anegative control, parental uninduced and untreated cells are shown inpanel 1. As a positive control, parental cells that were transfectedwith pMX-GFP plasmid encoding GFP protein are shown in panel 6. FIG. 15Cis a graph illustrating the correlation of GFP positive cells as afunction of Compound B02 concentration. To determine the effect ofCompound B02 on the efficiency of formation of GFP-positive cells(transfection plus GFP expression) 293 HEK cells were treated withCompound B02 in 5 μM, 10 μM, and 20 μM concentration and thentransfected with pMX-GFP-plasmid. Data are shown as a graph (denoted as“pMX-GFP”, efficiency of formation of GFP-positive cells in the cellswithout Compound B02 treatment was expressed as 100% formationsefficiency) along with the data from FIG. 15B (panels 2-5) thatdemonstrate the effect of Compound B02 on the formation GFPpositivecells resulted from DSB-induced homologous recombination (denoted“I-SecI”, formation of GFP-positive cells in no Compound B02 treatmentcells was expressed as 100%). Experiments were repeated at least threetimes; error bars represent standard deviation.

FIG. 16, comprising FIGS. 16A-16D, illustrates that finding thattreatment with Compound B02 does not affect the expression levels ofI-SceI and RAD51 in human cells. As illustrated in FIG. 16A, 293 HEKcells carrying the DR-GFP reporter were treated with Compound B02 inindicated concentrations or left untreated for 1 hr; and then pCbASecplasmid expressing I-SecI was transfected into the cells by GenDrill™transfection reagent. After incubation, during which Compound B02 waspresent, until cell confluence (˜64 h) cells were lysed and theexpression level of I-SceI restriction endonuclease containing the HAantigen was determined by western blotting using HA-Tag antibodies.Actin that was probed with specific antibodies was used as a quantitystandard. The data from FIG. 16A is illustrated as a graph in FIG. 16B.As illustrated in FIG. 16C, log-phase 293 HEK cells carrying the DR-GFPwere incubated either in the absence or presence of Compound B02 (20 μM)until cells reached confluence (˜24 h), the level of RAD51 expressionwas analyzed by western blotting using specific antibodies against RAD51. Purified RAD51 protein (56 ng) was used as a standard. The data fromFIG. 16C is illustrated as a graph in FIG. 16D. Experiments wererepeated at least three times; error bars represent standard deviation.

FIG. 17, comprising FIGS. 17A-17C, illustrates the finding that CompoundB02 increases the sensitivity of MEF cells to DNA damaging agents. MEF(FIG. 17A) or Tp53−/− MEF (FIG. 17B) cells were treated with myomycin C(MMC) or cis-dichlorodiamine platinum (II) (cisDDP) for 1 h in theabsence or presence of Compound B02 (5 μM). In FIG. 17C, MEF and Tp53−/− MEF cells were treated with Compound B02 in indicatedconcentrations. Experiments were repeated at least three times; errorbars represent standard deviation.

FIG. 18 is a graph illustrating the finding that cotreatment of MEFcells with a PARP-1 inhibitor AZD2281 and Compound B02 further increasescell sensitivity to DNA damaging agents. MEF cells were treated with MMS(∘), and then some fractions of these cells were additionally treatedwith Compound B02 (5 μM) (Δ), AZD2281 (1 μM) (∘), or with both CompoundB02 (5 μM) and AZD2281 (1 μM) (□). Experiments were repeated at leastthree times; error bars represent standard deviation.

FIG. 19, comprising FIGS. 19A-19F, illustrates the finding that CompoundB02 interacts with RAD51 and inhibits its activities. FIG. 19A depictsthe structure of Compound B02. FIG. 19B depicts the scheme of the DNAstrand exchange and branch migration assays. The asterisk denotes the³²P label. FIG. 18C depicts the effect of Compound B02 (10 to 100 μM) onthe DNA strand exchange activity of RAD51, FIG. 19D is a graphillustrating the yield of RAD51- and RecA-generated joint molecules(JM). FIG. 19E depicts the effect of Compound B02 (10 to 100 μM) on thebranch migration activity of RAD51. Lanes 1 and 11 represent JMs beforeand after 8 h-incubation in the absence RAD51, respectively. FIG. 19F isa graph illustrating the yield of the RAD51- and RecA-generated nickedcircle (NC) product. The extent of JM and NC formation in the absence ofCompound B02 was expressed as 100%; the actual extent was 32% and 15% ofJM (relative to linear dsDNA) and 21% and 63% of nicked circles forRAD51 and RecA (relative to JM-substrate), respectively. Controlscontaining no RAD51 are shown in lane 1. Experiments were repeated atleast three times; error bars represent standard deviation (S.D.).

FIG. 20, comprising FIGS. 20A-20C, illustrates the finding that CompoundB02 does not inhibit the DNA strand exchange and branch migrationpromoted by RecA. FIG. 20A illustrates the scheme of the DNA strandexchange and branch migration assays. The asterisk denotes the ³²Plabel. FIG. 20B illustrates the effect of Compound B02 on the DNA strandexchange activity of RecA. FIG. 20C illustrates the effect of CompoundB02 on the DNA branch migration activity of Rec A. Initial DNAsubstrates are shown in lane 1. Experiments were repeated at least threetimes; representative gels are shown.

FIG. 21 illustrates the finding that Compound B02 binds directly toRAD51. Compound B02 (50 μM) was injected onto a sensor chip to whichRAD51 or RecA had been immobilized. The running buffer S wassupplemented with ATP (100 μM). Responses to Compound B02 werenormalized to the theoretical maximum response of the surface (Rmax),assuming a 1:1 interaction. Experiments were repeated at least threetimes; error bars represent S.D.

FIG. 22, comprising FIGS. 22A-22D, illustrates the measurement ofCompound B02 binding to RAD51 using SPR. The SPR analysis was performedon a GLH high-capacity sensor chip (Blu-Rad, Hercules, Calif.) with ahigh density of immobilized RAD51 (14,000 RU) (FIG. 22A and 22B) or RecA(9,000 RU) (FIGS. 22C and 22D). Compound B02 at concentrations of 6.25,12.5, 25, and 50 μM in buffer S without ATP (FIGS. 22A and 22C) or withATP (100 μM) (FIGS. 22B and 22D) was injected to the chip. A chip withthe immobilized HIV-INL4-3 capsid protein served as a reference. Coloredlines indicate experimental data, whereas black lines indicate fittingto the simple 1:1 binding model using the ProteOn Manager Softwareversion 3.0 (Bio-Rad). When Compound B02 was injected over RAD51 inbuffer S containing ATP (100 μM), the data did not fit to the simplebinding model, probably due to a heterogeneity in the nucleotide bindingstates of the immobilized RAD51. For Compound B02 binding to RAD51 inthe absence of ATP, kinetic values are as follows:ka=4.5(±0.3)×10³M⁻1s⁻¹; kd=2.5(±0.3)×10⁻²s⁻1; Kd=5.6 μM. Experimentswere repeated at least three times; numbers in parentheses representS.D.

FIG. 23, comprising FIGS. 23A-23C, illustrates the finding that B02disrupts the RAD51 fuel formation. FIG. 23A illustrates HEK cells whichwere exposed to 0.5 Gy of IR in either the presence (50 μM)or absence ofB02. RAD51 foci were visualized by immunostaining using RAD51antibodies. Nuclei were counterstained with DAPI. Bars indicate 20 μm.FIG. 23B illustrates the fraction of foci-positive cells (the cells with≧1 focus), which was determined by counting at least 150 cells in eachexperiment. FIG. 23C illustrates the determination of the mean of focinumber per nucleus in focipositive cells by counting at least 50 cellsin each experiment. Experiments were repeated three times; error barsrepresent S.D.

FIG. 24, comprising FIGS. 24A-24F, illustrates the finding that B02increases cell sensitivity to DNA-damaging agents. FIGS. 24A and 24Billustrate the survival of MEF treated with cisplatin or MMC for 1 h inthe absence or presence of B02 (5 μM). FIG. 24C illustrates the effectof B02 on survival of MEF and Tp53−/− MEF, FIG. 24D illustrates theeffect of B02 (0 to 10 μM) and RAD51 siRNA on HEK cells' sensitivity tocisplatin. HEK cells were transfected with RAD51 siRNA and incubated 40h before treatment with cisplatin at indicated concentrations and B02 (5μM). FIG. 24E illustrates the effect of B02 incubation time with HEK oncell sensitivity to cisplatin. HEK cells were treated with B02 (5 μM)for 1 h followed by addition of cisplatin (16 μM) and incubation for 1h. Then cisplatin was removed and cells were incubated with B02 (5 μM)for the indicated times followed by media replacement and additionalincubation for 7-10 days. FIG. 24F illustrates the effect of AZD2281(0.01 μM) and B02 (5 μM) on MEF sensitivity to MMS. Experiments wererepeated at least three times; error bars represent S.D.

FIG. 25, comprising FIGS. 25A-25B, illustrates the finding that CompoundB02 increases the sensitivity of Tp53^(−/−) MEF to cisplatin and MMC.Tp53^(−/−) MEF were treated with cisplatin (FIG. 25A) or MMC (FIG. 25B)for 1 h in the absence or presence of B02 (5 μM). Experiments wererepeated at least three times; error bars represent S.D.

FIG. 26, comprising FIGS. 26A-26B, illustrates the effect of RAD51 siRNAon the expression level of RAD51. FIG. 26A illustrates the RAD51expression levels 24, 48, 72, and 96 h after SiRNA transfectiondetermined by Western blotting. The RAD51 protein level 48 h aftertransfection of HEK cells with scrambled siRNA (denoted as “sc siRNA”)was expressed as 100%. Purified RAD 51 (3.2 ng) was used as a marker.FIG. 26B is a graphical depiction of the results of FIG. 26A.

FIG. 27 illustrates the finding that Compound B02 inhibits HR bydisrupting RAD51 binding to DNA. During the repair of DNA double-strandbreaks, RAD51 binds to ssDNA forming the nucleoprotein filament. Thefilament searches for homologous dsDNA sequence to form joint molecules.The homologous DNA then is used as a template for DNA polymerase.Dissociation of the joint molecules and re-annealing of DNA ends lead tothe restoration of the DNA structure. Compound B02 inhibits HR bydisrupting formation of the RAD51-ssDNA filament and interaction of thefilament with dsDNA during the search for homology.

FIG. 28, comprising FIGS. 28A-28B, illustrates the finding that CompoundB02 does not affect RAD51 oligomerization. FIG. 28A illustrates theelution profiles of RAD51 (13.5 μM) that was either untreated (dashedcurve) or treated (solid curve) with B02 (200 μM) for 10 min at 37° C.prior to chromatography on a Superose 6 10/300 GL column (GEHealthcare). 0.3-ml fractions were collected. The void volume (7.5 ml)and the protein markers (thyroglobulin, 670 kDa; γ-globulin, 158 kDa;ovalbumin, 44 kDa; myoglobin, 17 kDa) are indicated. FIG. 28Billustrates the fractions analyzed by 12% SDS-PAGE. Prestained molecularweight markers and a RAD51 marker are shown in lanes 1 and 14,respectively.

DETAILED DESCRIPTION OF THE INVENTION

This invention includes the unexpected identification of novel selectivesmall-molecule RAD51 inhibitors and their utility in the treatment ofcancer. In order to identify selective RAD51 inhibitors, an efficienthigh throughput screening (HTS) of chemical compound libraries wasperformed using an assay based on fluorescence resonance energy transfer(FRET). Compounds found to inhibit RAD51 DNA strand exchange activitywere further analyzed by the D-loop assay (a secondary non-fluorescentDNA strand exchange assay), and potent RAD51 inhibitors were thenidentified.

Due to their unique mechanism, the compounds contemplated within theinvention are useful in overcoming the chemoresistance andradioresistance of human cancers. In one aspect, treatment of a subjectwith compounds contemplated within the invention enhances cellsensitivity to radiation treatments or chemotherapeutic agents, such asDNA cross-linking agents, cisplatin and mitomycin C. In another aspect,a subject treated with compounds contemplated within the invention alongwith a chemotherapeutic agent and/or radiation enjoys greater overallefficacy in the cancer treatment and/or prevention, as compared to theefficacy observed with the same dose of chemotherapeutic agent and/orradiation alone. In yet another aspect, a subject treated with compoundscontemplated within the invention may be treated with lower doses of thechemotherapeutic agent and/or radiation of choice, and still experiencesimilar efficacy in cancer treatment and/or prevention, as compared tothe standard dose of chemotherapeutic agent and/or radiation. This hasthe advantage of reducing complications due to toxicity from radiationtherapy or chemotherapy, and reducing recovery times for the subject.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures inbiochemisty, analytical chemistry and organic chemistry and thosewell-known and commonly employed in the art. Standard techniques ormodifications thereof are used for chemical syntheses and chemicalanalyses.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element. The term “about” will be understood by persons of ordinaryskill in the art and will vary to some extent on the context in which itis used. As used herein, “about” when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, the term “chemotherapeutic agent” or “chemotherapeuticagent” refers to a chemical compound, chemical conjugate, peptide,protein, antibody and the like that finds use in treating, preventing,or reducing the symptoms of cancer.

As used herein, the term “BHQ1” or “black hold quencher 1” refers to thefollowing moiety or a derivative (here, BHQ1 is illustrated as bound toa 5′-oligo through a phosphate bond):

As used herein, the term “EBVQ”, Compound B02 or Compound Ia areinterchangeable and refer to(E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one or a saltthereof.

An “amino acid” as used herein is meant to include both natural andsynthetic amino acids, and both D and L amino acids. “Standard aminoacid” means any of the twenty L-amino acids commonly found in naturallyoccurring peptides. “Nonstandard amino acid residues” means any aminoacid, other than the standard amino acids, regardless of whether it isprepared synthetically or derived from a natural source. As used herein,“synthetic amino acid” also encompasses chemically modified amino acids,including but not limited to salts, amino acid derivatives (such asamides), and substitutions. Amino acids contained within the peptides,and particularly at the carboxy- or amino-terminus, can be modified bymethylation, amidation, acetylation or substitution with other chemicalgroups which can change a peptide's circulating half-life withoutadversely affecting activity of the peptide. Additionally, a disulfidelinkage may be present or absent in the peptides.

As used herein, the terms “protein”, “peptide” and “polypeptide” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. The term “peptide bond”means a covalent amide linkage formed by loss of a molecule of waterbetween the carboxyl group of one amino acid and the amino group of asecond amino acid. A protein or peptide must contain at least two aminoacids, and no limitation is placed on the maximum number of amino acidsthat may comprise the sequence of a protein or peptide. Polypeptidesinclude any peptide or protein comprising two or more amino acids joinedto each other by peptide bonds. As used herein, the term refers to bothshort chains, which also commonly are referred to in the art aspeptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types. “Proteins” include, for example, biologicallyactive fragments, substantially homologous proteins, oligopeptides,homodimers, heterodimers, protein variants, modified proteins,derivatives, analogs, and fusion proteins, among others. The proteinsinclude natural proteins, recombinant proteins, synthetic proteins, or acombination thereof. A protein may be a receptor or a non-receptor.

As used herein, amino acids are represented by the full name thereof, bythe three-letter code, as well as the one-letter code correspondingthereto, as indicated in the following table. The structure of aminoacids and their abbreviations can also be found in the chemicalliterature, such as in Stryer, 1988, “Biochemistry”, 3^(rd) Ed., W. H.Freeman and Co., New York.

Three- One- Letter Letter Full Name Code Code Alanine Ala A Arginine ArgR Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Cystine Cys-CysC-C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

As used herein, the term “fragment,” as applied to a protein or peptide,refers to a subsequence of a larger protein or peptide. A “fragment” ofa protein or peptide may be at least about 20 amino acids in length; forexample at least about 50 amino acids in length; at least about 100amino acids in length, at least about 200 amino acids in length, atleast about 300 amino acids in length, and at least about 400 aminoacids in length (and any integer value in between).

By “nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone linkages, and combinations of suchlinkages. The term nucleic acid also specifically includes nucleic acidscomposed of bases other than the five biologically occurring bases(adenine, guanine, thymine, cytosine and uracil). The term “nucleicacid” typically refers to large polynucleotides.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

The term “RNA” as used herein is defined as ribonucleic acid.

The term “recombinant DNA” as used herein is defined as DNA produced byjoining pieces of DNA from different sources.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides, at least about 1000 nucleotides to about 1500nucleotides; or about 1500 nucleotides to about 2500 nucleotides; orabout 2500 nucleotides (and any integer value in between).

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “oligonucleotide” typically refers to short polynucleotides,generally no greater than about 60 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleotides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e., C₁₋₆means one to six carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropymethyl.

As used herein, the term “substituted alkyl” means alkyl, as definedabove, substituted by one, two or three substituents selected from thegroup consisting of halogen, —OH, alkoxy, —NH₂, —N(CH₃)₂, —C(═O)OH,trifluoromethyl, —C—N, —C(═O)O(C_(1-C) ₄)alkyl, —C(═O)NH₂, —SO₂NH₂,—C(═NH)NH₂, and —NO₂, preferably containing one or two substituentsselected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and—C(═O)OH, more preferably selected from halogen, alkoxy and —OH.Examples of substituted alkyls include, but are not limited to,2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃)alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quatomized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂OH, CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂—CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings)wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples includephenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, mostpreferred is phenyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one to three carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂-phenyl. Preferred is aryl-CH₂— and aryl-CH(CH₃)—.The term “substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkylfunctional group in which the aryl group is substituted. Preferred issubstituted aryl(CH₂)—. Similarly, the term “heteroaryl-(C₁C₃)alkyl”means a functional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. Preferred isheteroaryl-(CH₂)—. The term “substituted heteroaryl-(C₁-C₃)alkyl” meansa heteroaryl-(C₁C₃)alkyl functional group in which the heteroaryl groupis substituted. Preferred is substituted heteroaryl-(CH₂)—.

As used herein, the term “heterocycle” or “heterocyclyl” or“heterocyclic” by itself or as part of another substituent means, unlessotherwise stated, an unsubstituted or substituted, stable, mono- ormulti-cyclic heterocyclic ring system that consists of carbon atoms andat least one heteroatom selected from the group consisting of N, O, andS, and wherein the nitrogen and sulfur heteroatoms may be optionallyoxidized, and the nitrogen atom may be optionally quaternized. Theheterocyclic system may be attached, unless otherwise stated, at anyheteroatom or carbon atom that affords a stable structure. A heterocyclemay be aromatic or non-aromatic in nature. In one embodiment, theheterocycle is a heteroaryl.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includetetrahydroquinoline and 2,3-dihydrobenzoluryl.

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, imidazoline, pyrazolidine, dioxolane, sufolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin and hexamethyleneoxide.

Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl(particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl,pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl,pyrazolyl (particulary 3- and 5-pyrazolyl), isothiazolyl,1,2,3-trizolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiodiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles include indolyl (particularly 3-,4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl,isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particulary 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particulary 3-,4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benztriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

The aforementioned listing of heterocyclyl and heteroaryl moieties isintended to be representative and not limiting.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup.

For aryl, aryl-(C₁-C₃)alkyl and hetercyclyl groups, the term“substituted” as applied to the rings of these groups refers to anylevel of substitution, namely mono-, di-, tri-, tetra-, orpenta-substitution, where such substitution is permitted. Thesubstituents are independently selected, and substitution may be at anychemically accessibly position. In one embodiment, the substituents varyin number between one and four. In another embodiment, the substituentsvary in number between one and three. In yet another embodiment, thesubstituents vary in number between one and two. In yet anotherembodiment, the substituents vary in independently selected from thegroup consisting of C₁₋₆ alkyl, —OH, C₁₋₆ alkoxy, halo, amino, acetamidoand nitro. In yet another embodiment, the substituents are independentlyselected from the group consisting of C₁₋₆ alkyl, C₁₋₆ alkoxy group,acetamido, and nitro. As used herein, where a substituent is an alkyl oralkoxy group, the carbon chain may be branched, straight or cyclic, withstraight being preferred.

By the term “specifically binds,” as used herein, is meant a molecule,such as an antibody or a small molecule, which recognizes and binds toanother molecule or feature, but does not substantially recognize orbind other molecules or features in a sample.

The phrase “inhibit,” as used herein, means to reduce a molecule, areaction, an interaction, a gene, an mRNA, and/or a protein'sexpression, stability, function or activity by a measureable amount orto prevent entirely. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate a protein, a gene,and an mRNA stability, expression, function and activity, e.g.,antagonists.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result. Such results may include, butare not limited to, the treatment of a disease or condition asdetermined by any means suitable in the art.

As used herein, the term “pharmaceutical composition” refers to amixture of at least one compound of the invention with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to, intravenous, oral, acrosol,parenteral, ophthalmic, pulmonary and topical administration.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerins, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the term “salt” embraces addition salts of free acids orfree bases that are compounds useful within the invention. Suitable acidaddition salts may be prepared from an inorganic acid or from an organicacid. Examples of inorganic acids include hydrochloric, hydrobromic,hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric andtetrafluoroboronic acids. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes or organic acids, examples of whichinclude formic, acetic, propionic, succinic, glycolic, gluconic, lactic,malic, tartaric, citric, ascorbic, gluouronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzioc,phenylacetic, mandelic, embomic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanille,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable base addition salts ofcompounds useful within the invention include, for example, metallicsalts including alkali metal, alkaline earth metal and transition metalsalts such as, for example, lithium, calcium, magnesium, potassium,sodium and zinc salts. Acceptable base addition salts also includeorganic salts made from basic amines such as, for example,N,N′-debenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethyenediamine, meglumine (N-methyl-glucamine) and procaine. All ofthese salts may be prepared by conventional means from the correspondingfree base compound by reacting, for example, the appropriate acid orbase with the corresponding free base.

An “individual”, “patient” or “subject”, as that term is used therein,includes a member of any animal species including, but are not limitedto, birds, humans and other primates, and other mammals includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and dogs. Preferably, the subject is a human.

The term “treat” or “treating”, as used herein, means reducing thefrequency with which symptoms are experienced by a subject oradministering an agent or compound to reduce the frequency and/orseverity with which symptoms are experienced. As used herein,“alleviate” is used interchangeably with the term “treat.” Treating adisease, disorder or condition may or may not include completeeradication or elimination of the symptom. The term “therapeutic” asused herein means a treatment and/or prophylaxis. A therapeutic effectis obtained by suppression, remission, or eradication of UVR-inducedskin damage.

As used herein, the term “container” includes any receptacle for holdingthe pharmaceutical composition. For example, in one embodiment, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition ant theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating or preventing adisease in a subject.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositionand/or compound of the invention in a kit. The instructional material ofthe kit may, for example, be affixed to a container that contains thatcompound and/or composition of the invention or be shipped together witha container which contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublications or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Compounds Useful Within the Invention

In one aspect, the compound useful within the compositions and methodsof the invention is the compound of Formula (1):

wherein:

R¹ and R² independently H, C₁-C₆ alkyl, C₁-C₆ substituted alkyl, phenyl,substituted phenyl, heteroaryl, substituted heteroaryl, heterocycyl,substituted heterocyclyl, —(C₁-C₆)alkylene-phenyl,—(C₁-C₆)alkylene-substituted phenyl, —(C₁-C₆)alkylene-heteroaryl, or—(C₁-C₆)alkylene-substituted heteroaryl;

R³ is H, C₁-C₆ alkyl, O—(C₁-C₆ alkyl), F, Cl, Br or I; or a saltthereof.

In one embodiment, R¹ is H, methyl, phenyl, substituted phenyl,heteroaryl, substituted heteroaryl, —(C₁-C₆)alkylene-phenyl,—(C₁-C₆)alkylene-substituted phenyl, —(C₁-C₆)alkylene-heteroaryl, or—(C₁-C₆)alkylene-substituted heteroaryl. In another embodiment, R¹ isphenyl, substituted phenyl, heteroaryl, or substituted heteroaryl. Inyet another embodiment, R¹ is phenyl, o-tolyl, m-tolyl, p-tolyl,o-methoxyphenyl, m-methoxyphenyl, p-methoxyphenyl, o-halophenyl,m-halophenyl, p-halophenyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl,2-pyridinyl, 3-pyridinyl, or 4-pyridinyl.

In one embodiment, R² is H, C₁-C₆ alkyl, phenyl, substituted phenyl,heteroaryl, substituted heteronryl, —(C₁-C₆)alkylene-phenyl,—(C₁-C₆)alkylene-substituted phenyl, —(C₁-C₆)alkylene-heteroaryl, or—(C₁-C₆)alkylene-substituted heteroaryl. In another embodiment, R² isC₁-C₆ alkyl, phenyl, substituted phenyl, heteroaryl, substitutedheteroaryl, —(C₁-C₆)alkylene-phenyl, or —(C₁-C₆)alkylene-substitutedphenyl. In yet another embodiment, R² is methyl, ethyl, n-propyl,isopropyl, phenyl, o-tolyl, m-tolyl, p-tolyl, benzyl or substitutedbenzyl.

In one embodiment, R³ is H, C₁-C₆ alkyl, O(C₁-C₆ alkyl), F or Cl. Inanother embodiment, R³ is H, C₁-C₆ alkyl, or O(C₁-C₆ alkyl). In yetanother embodiment, R³ is H, methyl, ethyl, methoxy or ethoxy. In yetanother embodiment, R³ is H.

In one embodiment, the compound of Formula (1) is selected from thegroup consisting of:

-   (E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1a)

-   (E)-3-ethyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1b)

-   (E)-2-(2-(pyridin-3-yl)vinyl)-3-(m-tolyl)quinazolin-4(3H)-one (1c)

mixtures thereof and salts thereof.

In another aspect, the compound useful within the compositions andmethods of the invention is salazinic acid, also known as1,4,10-trihydroxy-5-(hydroxymethyl)-8-methyl-3,7-dihydro-1H-benzo[6,7][1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde,which has formula (2):

or a salt therof.

In yet another aspect, the compound useful within the compositions andmethods of the invention is stictic acid, also known as scopularic acidor1,4-dihydroxy-10-methoxy-5,8-dimethyl-3,7-dioxo-3,7-dihydro-1H-benzo[6,7][1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde,which has formula (3):

or a salt thereof.

Compounds useful within the methods of the invention may be synthesizedusing techniques well-known in the art of organic synthesis or obtainedfrom commercial sources.

Compositions of the Invention

In one aspect, the invention includes a pharmaceutical compositioncomprising a compound of Formula (1) and a pharmaceutically acceptablecarrier. In one embodiment, the pharmaceutical composition furthercomprises a chemotherapeutic agent. In another embodiment, the agent isselected from the group consisting of an alkylating agent,antimetabolite, anthracycline, plant alkaloid, plant terpenoid,topoisomerase inhibitor, and antineoplastic.

In another aspect, the invention includes a pharmaceutical compositioncomprising a compound of Formula (2), a chemotherapeutic agent, and apharmaceutically acceptable carrier.

In another aspect, the invention includes a pharmaceutical compositioncomprising a compound of Formula (3), a chemotherapeutic agent, and apharmaceutically acceptable carrier.

Salts of the Compounds of the Invention

The compounds described herein may form salts with acids or bases, andsuch salts are included in the present invention. In one embodiment, thesalts are pharmaceutically acceptable salts. The term “salts” embracesaddition salts of free acids or free bases that are compounds of theinvention. The term “pharmaceutically acceptable salt” refers to saltsthat possess toxicity profiles within a range that affords utility inpharmaceutical applications. Pharmaceutically unacceptable salts maynonetheless possess properties such as high crystallinity, which haveutility in the practice of the present invention, such as for exampleutility in process of synthesis, purification or formulation ofcompounds of the invention.

Suitable pharmaceutically acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hudroxybutyric,salicyclic, galactaric and galacturonic acid.

Examples of pharmaceutically unacceptable acid addition salts include,for example, perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include, for example, metallic salts including alkalimetal, alkaline earth metal and transition metal salts such as, forexample, calcium, magnesium, potassium, sodium and zinc salts.Pharmaceutically acceptable base addition salts also include organicsalts made from basic amines such as, for example,N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples ofpharmaceutically unacceptable base addition salts include lithium saltsand cyanate salts. All of these salts may be prepared from thecorresponding compound by reacting, for example, the appropriate acid orbase.

Methods of Invention

The invention includes a method of treating or preventing cancer in asubject in need thereof. The method comprises the step of administeringto the subject a pharmaceutical compound comprising a pharmaceuticallyacceptable carrier and a pharmaceutically effective amount of a compoundof Formula (1):

wherein

R¹ and R² are independently H, C₁-C₆ alkyl, C₁-C₆ substituted alkyl,phenyl, substituted phenyl, heteroaryl, substituted heteroaryl,heterocyclyl, substituted heterocyclyl, —(C₁-C₆)alkylene-phenyl,—(C₁-C₆)alkylene-substituted phenyl, —(C₁-C₆)alkylene-heteroaryl, or—(C₁-C₆)alkylene-substituted heteroaryl;

R³ is H, C₁-C₆ alkyl, O(C₁-C₆alkyl), F, Cl, Br or I; or a salt thereof.The method further comprises the step of administering to the subject atreatment selected from the group consisting of (i) radiation therapy,and (ii) a pharmaceutical composition comprising a pharmaceuticallyeffective amount of a chemotherapeutic agent, whereby treating orpreventing the cancer in the subject.

In one embodiment, the compound of Formula (1) is selected from thegroup consisting of(E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1a),(E)-3-ethyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1b),(E)-2-(2-(pyridin-3-yl)vinyl)-3-(m-tolyl)quinazolin-4(3H)-one (1c),mixtures thereof and salts thereof.

In one embodiment, administering of the compound of Formula (1) isperformed at least 24 hours prior to administering to the subject theradiation therapy or the chemotherapeutic agent. In another embodiment,administering of the compound of Formula (1) is performed at least 12hours prior to administering to the the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (1) is performed at least 6 hours prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (1) is performed at least 3 hours prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (1) is performed at least 1 hour prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, the compositioncomprising the compound of Formula (1) is co-administered to the subjectwith the radiation therapy or the composition comprising thechemotherapeutic agent. In yet another embodiment, the compound ofFormula (1) and the chemotherapeutic agent are co-formulated in apharmaceutical composition. In yet another embodiment, the subject is ahuman.

The invention also includes a method of treating or preventing cancer ina subject in need thereof. The method comprises the step ofadministering to the subject a pharmaceutical composition comprising apharmaceutically acceptable carrier and a pharmaceutically effectiveamount of a compound of Formula (2):

or a salt thereof. The method further comprises the step ofadministering to the subject a treatment selected from the groupconsisting of (i) radiation therapy, and (ii) a pharmaceuticalcomposition comprising a pharmaceutically effective amount of achemotherapeutic agent; whereby treating or preventing the cancer in thesubject.

In one embodiment, administering of the compound of Formula (2) isperformed at least 24 hours prior to administering to the subject theradiation therapy or the chemotherapeutic agent. In another embodiment,administering of the compound of Formula (2) is performed at least 12hours prior to administering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (2) is performed at least 6 hours prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (2) is performed at least 3 hours prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (2) is performed at least 1 hour prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, the compositioncomprising the compound of Formula (2) is co-administered to the subjectwith the radiation therapy or the composition comprising thechemotherapeutic agent. In yet another embodiment, the compound ofFormula (2) and the chemotherapeutic agent are co-formulated in apharmaceutical composition. In yet another embodiment, the subject is ahuman.

The invention further includes a method of treating or preventing cancerin a subject in need thereof. The method comprises the step ofadministering to the subject a pharmaceutical composition comprising apharmaceutically acceptable carrier and a pharmaceutically effectiveamount of a compound of Formula (3):

or a salt thereof. The method further comprises the step ofadministering to the subject a treatment selected from the groupconsisting of (i) radiation therapy, and (ii) a pharmaceuticalcomposition comprising a pharmaceutically effective amount of achemotherapeutic agent; whereby treating or preventing the cancer in thesubject.

In one embodiment, administering of the compound of Formula (3) isperformed at least 24 hours prior to administering to the subject theradiation therapy or the chemotherapeutic agent. In another embodiment,administering of the compound of Formula (3) is performed at least 12hours prior to administering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (3) is performed at least 6 hours prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (3) is performed at least 3 hours prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, administering of thecompound of Formula (3) is performed at least 1 hour prior toadministering to the subject the radiation therapy or thechemotherapeutic agent. In yet another embodiment, the compositioncomprising the compound of Formula (3) is co-administered to the subjectwith the radiation therapy or the composition comprising thechemotherapeutic agent. In yet another embodiment, the compound ofFormula (3) and the chemotherapeutic agent are co-formulated in apharmaceutical composition. In yet another embodiment, the subject is ahuman.

Combination Therapies

The compounds contemplated within the invention or salts thereof may beuseful in the methods of present invention in combination with radiationtherapy and/or a compound useful for treating cancer (generally referredto as “chemotherapeutic agent”). These additional compounds may comprisecompounds of the present invention or compounds (such as commerciallyavailable compounds) known to treat, prevent, or reduce the symptoms ofcancer. In one embodiment, the combination of a compound contemplatedwithin the invention and a chemotherapeutic agent has additive,complementary or synergistic effects in the treatment of cancer in asubject, or prevention of cancer in a subject. In another embodiment,the combination of a compound contemplated within the invention andradiation therapy has additive, complementary or synergistic effects inthe treatment of cancer in a subject, or prevention or cancer in asubject.

Radiation Therapy

In one aspect, a compound contemplated within the invention or a saltthereof may be used in combination with radiation therapy.

Radiation therapy, radiation oncology, or radiotherapy, sometimesabbreviated to XRT, is the medical use of ionizing radiation as part ofcancer treatment to control malignant cells. Radiotherapy may be used tocurative or adjuvant treatment. It is used as palliative treatment(where cure is not possible and the aim is for local disease control orsymptomatic relief) or as therapeutic treatment (where the therapy hassurvival benefit and it can be curative).

Radiotherapy is used for the treatment of malignant cancer, and may beused as a primary or adjuvant modality. It is also common to combineradiotherapy with surgery, chemotherapy, hormone therapy, immunotherapyor a mixture of the four. Most common cancer types can be treated withradiotherapy in some way. The precise treatment intent (curative,adjuvant, neoadjuvant, therapeutic, or palliative) will depend on thetumor type, location, and stage, as well as the general health of thepatient.

Radiation therapy is commonly applied to the cancerous tumor. Theradiation fields may also include the draining lymph nodes if they areclinically or radiologically involved with tumor, or if there is thoughtto be a risk of subclinical malignant spread. Brachytherapy, in which aradiation source is placed inside or next to the area requiringtreatment, is another form of radiation therapy that minimizes exposureto healthy tissue during procedures to treat cancers of the breast,prostate and other organs.

The amount of radiation used in photon radiation therapy is measured ingray (Gy), and varies depending on the type and stage of cancer beingtreated. For curative cases, the typical dose for a solid epithelialtumor ranges from 60 to 80 Gy, while lymphomas are treated with 20 to 40Gy in 1.8-2 Gy fractions (for breast, head, and neck cancers.) Manyother factors are considered by radiation oncologists when selecting adose, including whether the patient is receiving chemotherapy, patientcomorbidities, whether radiation therapy is being administered before orafter surgery, and the degree of success of surgery.

Chemotherapeutic Agents

In one aspect of the invention, a compound of the invention or a saltthereof may be used in combination with a chemotherapeutic agent.

In one embodiment, a compound of the invention is co-administered with achemotherapeutic agent to the subject in need thereof. In anotherembodiment, a compound of the invention and a chemotherapeutic agent areadministered to the subject as part of the same pharmaceuticalformulation. In yet another embodiment, a compound of the invention anda chemotherapeutic agent are administered separately to the subject inneed thereof.

Most of the approved chemotherapeutic agents may be divided intoalkylating agents, antimetabolites, anthracyclines, plant alkaloids andterpenoids, topoisomerase inhibitors, antineoplastics and otherantitumour agents. These drugs affect cell division or DNA synthesis andfunction directly or indirectly.

Some newer chemotherapeutic agents do not directly interfere with DNAsynthesis and function. These include monoclonal antibodies and tyrosinekinase inhibitors e.g. imatinib mesylate (Gleevec or Glivec), whichdirectly targets a molecular abnormality in certain types of cancer(chronic myelogenous leukemia, gastrointestinal stromal tumors), and aregenerally referred to as targeted therapies.

In addition, some drugs that modulate tumor cell behavior withoutdirectly attacking those cells, such as hormones, may be used in thetreatment of cancer.

Non-limiting examples of chemotherapeutic agents are provided below.

Alkylating Agents:

Alkylating agents alkylate nucleophilic functional groups present incells. They impair cell function by forming covalent bonds with theamino, carboxyl, sullhydryl, and phosphate groups in biologicallyimportant molecules, and in particular by chemically modifying a cell'sDNA. Cisplatin, carboplatin, oxaliplatin, mechlorethamine,cyclophosphamide, chlorambucil, and ifosfamide are alkylating agents.

Anti-Metabolites:

Anti-metabolites masquerade as purines (azathioprine, mercaptopurine) orpyrimidines, which are building blocks of DNA. By competing outnaturally occurring purines or pyrimidines, anti-metabolites preventthese building blocks from becoming incorporated into DNA during the “S”phase (of the cell cycle), thus stopping normal development anddivision. Anti-metabolites also affect RNA synthesis. Due to theirefficiency, anti-metabolites are the most widely used cytostatics.

Plant Alkaloids and Terpenoids:

These plant alkaloids block cell division by preventing microtubulefunction. Microtubules are vital for cell division, and, without them,cell division cannot occur. The main examples of plant alkaloids arevinca alkaloids and taxanes.

(a) Vinca Alkaloids

Vinca alkaloids bind to specific sites on tubulin, inhibiting assemblyof tubulin into microtubules (M phase of the cell cycle). The vincaalkaloids include vincristine, vinblastine, vinorelbine and vindesine.

(b) Podophyllotoxin

Podophyllotoxin is a plant-derived compound said to help with digestionand used to produce two other cytostatic drugs, etoposide andteniposide. They prevent the cell from entering the GI phase (the startof DNA replication) and the replication of DNA (the S phase). The exactmechanism of its action is unknown. The substance has been primarilyobtained from the American Mayapple (Podophyllum peltatum). Recently ithas been discovered that a rare Himalayan Mayapple (Podophyllumhexandrum) contains it in a much greater quantity, but, as the plant isendangered, its supply is limited. Studies have been conducted toisolate the genes involved in the substances's production, so that itcould be obtained recombinantly.

(c) Taxanes

The prototype taxane is the natural product paclitaxel, originally knownas Taxol and first derived from the bark of the Pacific Yew tree.Ducetaxel is a semi-synthetic analogue of paclitaxel. Taxanes enhancestability of microtubules, preventing the separation of chromosomesduring anaphase.

Topoisomerase Inhibitors:

Topoisomerase are essential enzymes that maintain the topology of DNA.Inhibition of type I or type II topoisomerases interferes with bothtranscription and replication of DNA by upsetting proper DNAsupercoiling. Type I topoisomerase inhibitors include camptothecins:irinotecan and topotecan. Type II inhibitors include amsacrine,etoposide, etoposide phosphate, and teniposide. These are semisyntheticderivatives of epipodephylltoxins, alkaloids naturally occurring in theroot of American Mayapple (Podophyllum peltatum).

Antineoplastics:

These include the immunosuppressant dactinomycin (which is used inkidney transplantations), doxorubicia, epirubicin, bleomycin and others.

Anticancer agents working through different cytotoxic mechanisms mayalso be combined in “chemotherapy regimens” in order to target aspecific type of cancer. Chemotherapy regimens are often identified byacronyms, identifying the agents used in combination. However, theletters used are not consistent across regimens, and in some cases (forexample, “BEACOPP”), the same letter combination is used to representtwo different treatments. Non-limiting examples of combinations used inclinical settings are listed below, in terms of acronyms, compositionsand cancer types:

-   -   ABVD: Adriamycin (doxorubicin), bleomycin, vinblastine,        dacarbazine—Hodgkins's lymphoma    -   AC: Adriamycin (doxorubicin), cyclophosphamide—Breast cancer    -   BEACOPP: Bleomycin, etoposide, Adriamycin (doxorubicin),        cyclophosphamide, Oncovin (vincristine), procarbazine,        prednisone—Hodgkin's lymphoma    -   BEP: Bleomycin, etoposide, platinum agent (cisplatin)—Testicular        cancer, germ cell tumors    -   CA: Cyclophosphamide, Adriamycin (doxorabicin) (same as        AC)—Breast cancer    -   CAF: Cyclophosphamide, Adriamycin (doxorubicin), fluorouracil        (5-FU)—Breast cancer    -   CAV: Cyclophosphamide, Adriamycin (doxorubicin),        vincristine—Lung cancer    -   CBV: Cyclophosphamide, BCNU (carmustine), VP-16        (etoposide)—Lymphoma    -   ChIVPP/EVA: Chlorambucil, vincristine (Oncovin), procarbazine,        prednisone, etoposide, vinblastine, Adriamycin        (doxorubicin)—Hodgkin's lymphoma    -   CHOP: Cyclophosphamide, hydroxydoxorabicin (doxorubicin),        vincristine (Oncovin), prednisone—Non-Hodgkin lymphoma    -   CHOP-R or R-CHOP: CHOP—rituximab—B cell non-Hodgkin lymphoma    -   COP or CVP: Cyclophosphamide, Oncovin (vincristine),        prednisone—Non-Hodgkin lymphoma in patients with history of        cardiovascular disease    -   CMP: Cyclophosphamide, methotrexate, fluorouracil (5-FU)—Breast        cancer    -   COPP: Cyclophosphamide, Oncovin (vincristine), procarbazine,        prednisone—Non-Hodgkin lymphoma    -   EC: Epirubicin, cyclophosphamide—Breast cancer    -   ECF: Epirubicin, cisplatin, fluorouracil (5-FU)—Gastric cancer        and oesophageal cancer    -   EP: Etoposide, platinum agent (cisplatin)—Testicular cancer,        germ cell tumors    -   EPOCH: Etoposide, prednisone, Oncovin, cyclophosphamide, and        hydroxydaunorubicin—Lymphomas    -   FEC: Fluorouracil (5-FU), epirubicin, cyclophosphamide—Breast        cancer    -   FL (Also know as Mayo): Fluorouracil (5FU), leucovocin (folinic        acid)—Colorectal cancer    -   FOLFOX: Fluorouracil (5-FU), leucovorin (folimic acid),        oxaliplatin—Colorectal cancer    -   FOLFIRI: Fluorouracil (5-FU), leucovorin (folimic acid),        irinotecan—Colorectal cancer    -   ICE: ifosfamide, carboplatin, etoposide (VP-16)—Aggressive        lymphomas, progressive neuroblastoma    -   ICE-R: ICE+rituximab—High-risk progressive or recurrent        lymphomas    -   m-BACOD: Methotrexate, bleomycin, Adriamycin (doxorubicin),        cyclophosphamide, Oncovin (vincristine),        dexamethasone—Non-Hodgkin lymphoma    -   MACOP-B: Methotrexate, leucovorin (folinic acid), Adriamycin        (doxorubicin), cyclophosphamide, Oncovin (vincristine),        prednisone, bleomycin—Non-Hodgkin lymphoma

MOPP: Mechlorethamine, Oncovin (vincristine), procarbazine,prednisone—Hodgkin's lymphoma

-   -   MVAC: methotrexate, vinblastine, adriamycin, cisplatin—Advanced        bladder cancer[2]    -   PCV: Procarbazine, CCNU (lomustine), vincristine—Brain tumors    -   ProMACE-MOPP: Methotrexate, Adriamycin (doxorubicin),        cyclophosphamide, etoposide+MOPP—Non-Hodgkin lymphoma    -   ProMACE-CytaBOM: Prenisone, doxorubicin (adriamycin),        cyclophosphamide, etoposide, cytarabine, bleomycin, Oncovin        (vincristine), methotrexate, leucovorin—Non-Hodgkin lymphoma    -   R-FCM: Rituximab, fludarabine, cyclophosphamide, mitoxantrone—B        cell non-Hodgkin lymphoma    -   Stanford V: Doxorubicin, mechlorethamine, bleomycin,        vinblastine, vincristine, etoposide, prednisone—Hodgkin's        lymphoma    -   Thal/Dex: Thalidomide, dexamethasone—Multiple myeloma    -   TIP: Paclitaxel, ifosfamide, platinum agent cisplatin—Testicular        cancer, germ cell tumors in salvage therapy    -   VAC: Vincristine, Actinomycin, Cyclophasphamide—Rhabdomyosarcoma    -   VAD: Vincristine, Actinomycin (doxorubicin),        dexamethasone—Multiple myeloma    -   VAPEC-B: Vincristine, Actinomycin (doxorubicin), prednisone,        etoposide, cyclophosphamide, bleomycin—Hodgkin's lymphoma    -   VIP: Etoposide, ifosfamide, platinum agent cisplatin—Testicular        cancer, germ cell tumors

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the media-effect equation (Chou & Talulay, 1984, Adv.Enzyme Regul. 22: 27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Pharmaceutical Compositions and Therapies

Administration of a compound useful within the invention may be achievedin a number of different ways, using methods known in the art. Thetherapeutic and prophylactic methods of the invention thus encompass theuse of pharmaceutical compositions comprising the compounds usefulwithin the invention to practice the methods of the invention. Thepharmaceutical compositions useful for practicing the invention may beadministered to deliver a dose of 1 ng/kg/day to 100 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in the pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Although the description of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commericallyrelevant mammals such as non-human primates, cattle, pigs, horses,sheep, cats, and dogs.

Typically, dosages which may be administered in a method of theinvention to an animal, preferably a human, range in amount from 0.5 μgto about 50 mg per kilogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration, the dosage of the compound will preferably vary fromabout 1 μg to about 10 mg per kilogram of body weight of the animal.More preferably, the dosage will vary from about 3 μg to about 1 mg perkilogram of body weight of the animal.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, parenteral, topical, buccal, or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a pharmaceuticallyacceptable carrier or one or more other accessory ingredients, and then,if necessary or desirable, shaping or packaging the product into adesire single- or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged,or solid in bulk, as a single unit does, or as a plurality of singleunit doses. As used herein, a “unit does” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient that would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound or conjugateof the invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers that are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mark Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the ease of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,in the composition. Prolonged absorption of the injectable compositionsmay be brought about by including in the composition an agent thatdelays absorption, for example, aluminum monostearate or gelatin. In oneembodiment, the pharmaceutically acceptable carrier is not DMSO alone.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” that may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton Pa.), which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombination thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

The composition preferably includes an anti-oxidant and a chelatingagent that inhibits the degradation of the compound. Preferredantioxidants for some compounds are BHT, BHA, alpha-tocopherol andascorbic acid in the preferred range of about 0.01% to 0.3% and morepreferably BHT in the range of 0.03% to 0.01% by weight by total weightof the composition. Preferably, the chelating agent is present in anamount of from 0.01% to 0.5% by weight by total weight of thecomposition. Particularly preferred chelating agents include edetatesalts (e.g. disodium edetate) and citric acid in the weight range ofabout 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10%by weight by total weight of the composition. The chelating agent isuseful for chelating metal ions in the composition that may bedetrimental to the shelf life of the formulation. While BHT and disodiumedetate are the particularly preferred antioxidant and chelating agentrespectively for some compounds, other suitable and equivalentantioxidants and chelating agents may be substituted therefore as wouldbe known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demalcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Knows suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragaeanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxyproplmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one which comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspension, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Controlled- or sustained-release formulations of a composition of theinvention may be made using conventional technology, in addition to thedisclosure set forth elsewhere herein. In some cases, the dosage formsto be used can be provided as slow or controlled-release of one or moreactive ingredients therein using, for example, hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmoticsystems, multilayer coatings, microparticles, liposomes, or microspheresor a combination thereof to provide the desired release profile invarying proportions. Suitable controlled-release formulations known tothose of ordinary skill in the art, including those described herein,can be readily selected for use with the compositions of the invention.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, nanoparticles,or microspheres or a combination thereof that facilities thecontrolled-release of the active ingredient.

Administration/Dosing

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after a diagnosis of disease. Further, severaldivided dosages, as well as staggered dosages may be administered dailyor sequentially, or the dose may be continuously infused, or may bebolus injection. Further, the dosages of the therapeutic formulationsmay be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to asubject, preferably a mammal, more preferably a human, may be carrierout using known procedures, at dosages and for periods of time effectiveto prevent or treat disease. An effective amount of the therapeuticcompound necessary to achieve a therapeutic effect may vary according tofactors such as the activity of the particular compound employed; thetime of administration; the rate of excretion of the compound; theduration of the treatment; other drugs, compounds or materials used incombination with the compound; the state of the disease or disorder,age, sex, weight, condition, general health and prior medical history ofthe subject being treated, and like factors well-known in the medicalarts. Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation. A non-limiting example of an effectivedose range for a therapeutic compound of the invention is from about 1and 5,000 mg/kg of body weight/per day. One of ordinary skill in the artwould be able to study the relevant factors and make the determinationregarding the effective amount of the therapeutic compound without undueexperimentation.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc. The formulations of the pharmaceuticalcompositions described herein may be prepared by any method known orhereafter developed in the art of pharmacology. In general, suchpreparatory methods include the step of bringing the active ingredientinto association with a carrier or one or more other accessoryingredients, and then, if necessary or desirable, shaping or packagingthe product into a desired single- or multi-dose unit.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease in a subject.

In one embodiment, the compositions of the invention are administered tothe subject in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the subject in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary from subjectto subject depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any subject will be determined by the attendingphysical taking all other factors about the subject into account.

Compounds of the invention for administration may be in the range offrom about 0.1 mg to about 1,000 mg, about 0.2 mg to about 950 mg, about0.4 mg to about 900 mg, about 1 mg to about 850 mg, about 5 mg to about750 mg, about 20 mg to about 700 mg, about 30 mg to about 600 mg, about50 mg to about 500 mg, about 75 mg to about 400 mg, about 100 mg toabout 300 mg, about 120 mg to about 250 mg, and any and all whole orpartial increments therebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., a drug used fortreating the same or another disease as that treated by the compositionof the invention) as described herein is less than about 1,000 mg, orless than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 400 mg, or less than about 300 mg, or lessthan about 200 mg, or less than about 100 mg, or less than about 50 mg,or less than about 40 mg, or less than about 30 mg, or less than about25 mg, or less than about 20 mg, or less than about 15 mg, or less thanabout 10 mg, or less than about 5 mg, or less than about 2 mg, or lessthan about 1 mg, or less than about 0.5 mg, and any and all whole orpartial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a composition of the invention,alone or in combination with a second pharmaceutical agent; andinstructions for using the composition to treat, prevent, or reduce oneor more symptoms of a disease in a subject.

Routes of Administration

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, parenteral, sublingual, transdermal,transmucousal (e.g., sublingual, lingual, (trans)buccal,(trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal,and (trans)rectal), intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topicaladministration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, a paste, a gel, toothpaste, amouthwash, a coating, an oral rinse, or an emulsion. The compositionsintended for oral use may be prepared according to any method known inthe art and such compositions may contain one or more agents selectedfrom the group consisting of inert, non-toxic pharmaceuticallyexcipients that are suitable for the manufacture of tablets. Suchexcipients include, for example an inert diluent such as lactose;granulating and disintegrating agents such as cornstarch; binding agentssuch as starch; and lubricating agents such as magnesium stearate.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way or example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmoticallycontrolled release tablets. Tablets may further comprise a sweeteningagent, a flavoring agent, a coloring agent, a preservative, or somecombination of these in order to provide for pharmaceutically elegantand palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the composition of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents; fillers;lubricants; disintegrates; or wetting agents. If desired, the tabletsmay be coated using suitable methods and coating materials such asOPADRY™ film coating systems available from Colorcon, West Point, Pa.(e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, AqueousEnteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400).

Liquid preparation for oral administration may be in the form ofsolutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propylpara-hydroxy benzoates or sorbic acid). Liquid formulations of apharmaceutical composition of the invention which are suitable for oraladministration may be prepared, packaged, and sold either in liquid formor in the form of a dry product intended for reconstitution with wateror another suitable vehicle prior to use.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolldone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wad-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) will melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of a disease. Using a wax/pH-sensitive polymermix, a gastric insoluble composition may be obtained in which the activeingredient is entrapped, ensuring its delayed release.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, intraocular,intravitreal, subcutaneous, intraperitoneal, intramuscular, intrastemalinjection, intralumural, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulations thatare useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Topical Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for topical administration. There areseveral advantages to delivering compounds, including drugs or othertherapeutic agents, into the skin (dermal drug delivery) or into thebody through the skin (transdermal drug delivery). Transdermal compounddelivery offers an attractive alternative to injections and oralmedications. Dermal compound delivery offers an efficient way to delivera compound to the skin of a mammal, and preferably a human, and providesa method of treatment of the skin, or otherwise provides a method ofaffecting the skin, without the need to break or damage the outer layerof the skin. In the present invention, dermal delivery, by way or adermally-acting compound of the invention, provides these advantages fortreatment of a skin-related condition, disorder or disease.

A number of compounds, including some drugs, will penetrate the skineffectively simply because the molecules are relatively small and potentat small doses of 0.1 mg to 15 mg/day (Kanikkannan et al., 2000, Curr.Med. Chem. 7:593-608). Many other compounds and drugs can be deliveredonly when an additional enhancement system is provided to “force” themto pass through the skin. Among several methods of transdermal drubdelivery are electroporation, sonophoresis, iontophorsesis, permeationenhancers (cyclodextrins), and liposomes. While the aforementionedmethods are also included in the present invention for dermal deliveryof the compounds of the invention, liposomes represent a preferreddermal delivery method.

The composition of the invention may consist of the active ingredientalone, in a form suitable for administration to a subject, or thecomposition may comprise at least one active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the composition in the form of a physiologically acceptableester or salt, such as in combination with a physiologically acceptablecation or anion, as is well known in the art. Compositions of theinvention will also be understood to encompass pharmaceuticalcompositions useful for treatment of other conditions, disorders anddiseases associated with the skin.

In one aspect, a dermal delivery vehicle of the invention is acomposition comprising at least one first compound that can facilitatedermal delivery of at least one second compound associated with, or inclose physical proximity to, the composition comprising the firstcompound. As will be understood by the skilled artisan, when armed withthe disclosure set forth herein, such delivery vehicles include, butshould not be limited to, liposomes, nanosomes, phospholipid-basednon-liposome compositions (eg., selected cochleates), among others.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 0.001% to about 90% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

In one aspect of the invention, a dermal delivery system includes aliposome delivery system, and that the present invention should not beconstrued to be limited to any particular liposome delivery system.Based on the disclosure set forth herein, the skilled artisan willunderstand how to identify a liposome delivery system as being useful inthe present invention.

The present invention also encompasses the improvement of dermal andtransdermal drug delivery through the use of penetration enhancers (alsocalled sorption promoters or accelerants), which penetrate into skin toreversibly decrease the barrier resistance. Many compounds are known inthe art for penetration enhancing activity, including sulphoxides (suchas dimethylsulphoxide, DMSO), azones (e.g. laurocapram), pyrrolidones(for example 2-pyrrolidone, 2P), alcohols and alkanols (ethanol, ordecanol), glycols (for example propylene glycol, PG, a common excipientin topically applied dosage forms), surfactants (also common in dosageforms) and terpenes. Other enhancers include oleic acid, oleyl alcohol,ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide,polar lipids, or N-methyl-2-pyrrolidone.

In alternative embodiments, the topically active pharmaceutical orcosmetic composition may be optionally combined with other ingredientssuch as moisturizers, cosmetic adjuvants, anti-oxidants, chelatingagents, surfactants, foaming agents, conditioners, humectants, wettingagents, emulsifying agents, fragrances, viscosifiers, buffering agents,preservatives, sunscreens and the like. In another embodiment, apermeation or penetration enhancer is included in the composition and iseffective in improving the percutaneous penetration of the activeingredient into and through the stratum corneum with respect to acomposition lacking the permeation enhancer. Various permeationenhancers, including oleic acid, oleyl alcohol, ethoxydiglycol,laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, orN-methyl-2-pyrrolidone, are known to those of skill in the art.

In another aspect, the composition may further comprise a hydrotropicagent, which functions to increase disorder in the structure of thestratum corneum, and thus allows increased transport across the stratumcorneum. Various hydrotropic agents such as isopropyl alcohol, propyleneglycol, or sodium xylene sulfonate, are known to those of skill in theart. The compositions of this invention may also contain active amountsof retinoids (i.e., compounds that bind to any members of the family ofretinoid receptors), including, for example, tretinoin, retinol, estersof tretinoin and/or retinol and the like.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of an aqueous gel because ofrepeated patient use when it is exposed to contaminants in theenvironment from, for example, exposure to air or the patient's skin,including contact with the fingers used for applying a composition ofthe invention such as a therapeutic gel or cream. Examples ofpreservatives useful in accordance with the invention included but arenot limited to those selected from the group consisting of benzylalcohol, sorbic acid, parabens, imidurea and combinations thereof. Aparticularly preferred preservative is a combination of about 0.5% to2.0% benzyl alcohol and 0.05% sorbic acid.

The composition preferably includes an antioxidant and a chelating agentwhich inhibit the degradation of the compound for use in the inventionin the aqueous gel formulation. Preferred antioxidants for somecompounds are BHT, BHA, alpha-tocopherol and ascorbic acid in thepreferred range of about 0.01% to 5% and BHT in the range of 0.01% to 1%by weight by total weight of the composition. Preferably, the chelatingagent is present in an amount of from 0.01% to 0.5% by weight by totalweight of the composition. Particularly preferred chelating agentsinclude edetate salts (e.g., disodium edetate) and citric acid in theweight range of about 0.01% to 0.20% and more preferably in the range of0.02% to 0.10% by weight by total weight of the composition. Thechelating agent is useful for chelating metal ions in the compositionwhich may be detrimental to the shelf life of the formulation. While BHTand disodium edetate are the particularly preferred antioxidant andchelating agent respectively for some compounds, other suitable andequivalent antioxidants and chelating agents may be substitutedtherefore as would be known to those skilled in the art.

Additional components may include, but should not be limited to thoseincluding water, oil (eg., olive oil/PEG7), biovera oil, wax (eg.,jojoba wax), squalene, myristate (eg., isopropyl myristate),triglycerides (eg., captylic triglyceride), Solulan 98, cocoa butter,shea butter, alcohol (eg., behenyl alcohol), stearate (eg.,glycerol-monostearate), chelating agents (eg., EDTA), propylene gylcolSEPIGEL (Seppic, Inc., Fairfield, N.J.), silicone and siliconederivatives (eg., dimethicone, cyclomethicone), vitamins (eg., vitaminE), among others.

Buccal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

Rectal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants, andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, and preservatives.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837 and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041, WO03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

Controlled- or sustained-release formulations of a pharmaceuticalcompositions of the invention may be made using conventional technology,using for example proteins equipped with pH sensitive domains orprotease-cleavable fragments. In some cases, the dosage forms to be usedcan be provided as slow or controlled-release of one or more activeingredients therein using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, micro-particles, liposomes, or microspheres or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled-release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the pharmaceutical compositions of theinvention. Thus, single unit dosage forms suitable for oraladministration, such as tablets, capsules, gel-caps, and caplets, whichare adapted for controlled-release are encompassed by the presentinvention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased subject compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilities the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulations that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material that provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In a preferred embodiment of the invention, the compounds of theinvention are administered to a subject, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Kits of the Invention

The invention also includes a kit comprising a compound useful withinthe methods of the invention and an instructional material thatdescribes, for instance, administering the compound to a subject as aprophylactic or therapeutic treatment for cancer as described elsewhereherein. In one embodiment, the kit further comprises a (preferablysterile) pharmaceutically acceptable carrier suitable for dissolving orsuspending the therapeutic composition, comprising the compound usefulwithin the methods of the invention, for instance, prior toadministering the molecule to a subject. Optionally, the kit comprisesan applicator for administering the compound.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

The materials and methods employed in the experiments and the results ofthe experiments presented in this Example are now described.

Proteins and DNA:

RecA was purchased from USB Inc. (Cleveland, Ohio). Human RAD51 andRAD54 were purified as described (Sigurdsson et al., 2001, J. Biol.Chem. 276:8798-8806; Mazina & Mazin, 2004, J. Biol. Chem. 279:52042-51).The oligonucleotides used in this study were purchased from IDT Inc.(San Diego, Calif.) in a desalted form.

Oligonucleotide and pUC19 DNA substrates were prepared as described(Bugreev et al., 2006, Nature Protocols, published online Sep. 1, 2006;Rossi et al, Methods 51:336-46). ΦX174 ssDNA and dsDNA were purchasedfrom New England Biolabs (Ipswich, Mass.) and Invitrogen (Carlsbad,Calif.), respectively. The DNA concentrations are expressed as moles ofnucleotide.

(E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one) (CompoundB02) was purchased from Ryan Scientific Inc. (Mt. Pleasant, S.C.). Poly[ADP-ribose]polymerase 1 (PARP1) inhibitor AZD2281 (olaparib or4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fluorophenyl]methyl(2H)phthalazin-I-one) waspurchased from Selleck Chemicals LLC (Houston, Tex.).Cis-dichlorodiamine platinum (II) (cisDDP) and Mitomycin C (MMC) werepurchased from Sigma-Aldrich (St. Louis, Mo.). Gapped DNA was preparedby annealing the pBSK (1) XhoI-AlwNI fragment (2065 bp) to pBSK (+)ssDNA and purified as described previously.

Cell Culture:

Mouse embryonic fibroblasts (MEF) and Tp53−/−MEF cells are gifts fromDr. Astrinidis (Rajesh et al., 2010, DNA Repair (Arnst) 9, 458-67;Matthew et al., E. M., 2009, Cell Cycle 8, 4168-75). 293 human embryonickidney (HEK) cells with intrachromosomally based GFP (DRGFP) reportersystem (Pierce et al, 1999, Genes Dev. 13:2633-38), as well as pCBASSce(Pierce et al., 1999, Genes Dev. 13:2633-38) and pMX-GFP (Cell Biolabs,Inc. San Diego, Calif.) plasmids are gifts from Dr. Clifford.

Except otherwise indicated, all cell lines were maintained in Dalbecco'sModified Eagle medium (DMEM) (Sigma-Aldrich, St. Louis, Mo.)supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad,Calif.), 100 units/ml penicillin and 100 μg/ml streptomycin(Sigma-Aldrich, St. Louis, Mo.) in the presence of 5% CO₂ at 37° C.

Compound Libraries:

The NIH Small Molecule Repository (202,556 compounds) was used for theprimary screening for RAD51 inhibitors. All the compounds were dissolvedin DMSO (Sigma-Aldrich, St. Louis, Mo.); concentration of stocksolutions were 2.5 mM or 5 mM. In the working solutions the DMSOconcentration added with the stock of compounds was 2% (v/v), unlessindicated otherwise. The compounds for SAR analysis were purchased fromChembridge Co. (San Diego, Calif.).

Fluorescence-Based Strand Exchange Assay:

A fluorescence-based assay was developed to measure the RAD51 DNA strandexchange activity. In this assay, dsDNA substrate was prepared byannealing two complementary ssDNA oligonucleotides Oligo 25-FLU(fluorescein-25-FLU contains the fluorescein group, a donor fluorophorewith the excitation maximum at 490 nm and the emission maximum at 521nm, at the 5′-end. Oligo 26-BHQ1 contains the black hole quencher I(BHQI), a non-fluorescent acceptor, at the 3′-end. To form thenucleoprotein filament, RAD51 (200 nM) was incubated with a 48-mer ssDNA(Oligo 25; SEQ ID NO:2) (600 nM, nt) in DNA strand exchange buffercontaining 40 mM Tris-HCl (pH 7.8), 2 mM ATP, 5 mM CaCl₂, 1 mM DTT and100 μg ml⁻¹ BSA for 15 min at 37° C. DNA strand exchange was initiatedby addition of dsDNA (600 nM, nt) (Oligo 25-FLU: fluorescein-SEQ IDNO:3; Oligo 26-BHQI; SEQ ID NO:4-Black Hole Quencher I). Ca²⁺ stronglystimulates DNA strand exchange activity of RAD51, but not that of theyeast and bacterial RAD51 homologues (Bugree & Mazin, 2004, Proc. Natl.Acad. Sci. 101:9988-93). The reactions were carried out for indicatedperiod of time at 37° C. or 23° C., as indicated. The fluorescenceintensity was measured using a Fluoromax3 fluorimeter (Jobin Yvon,Edison, N.J.).

HTS of the NIH SMR for RAD51 Inhibitors:

To form the nucleoprotein filament, RAD51 (300 nM) was incubated with a48-mer ssDNA (SEQ ID NO:2) (600 nM, nt) in DNA strand exchange buffercontaining 40 mM HEPES (pH 7.8), 2 mM ATP, 5 mM CaCl₂, 1 mM DTT and 100μg ml⁻¹ BSA for 15 min at 37° C. 10 μl aliquots of the mixtures wereadded to the plates containing the NIH Small Molecule Repository andfurther incubated at 23° C. for 30 min. DNA strand exchange reactionswere initiated by addition of dsDNA (300 nM, nt) (fluorescein-SEQ IDNO:3 and SEQ ID NO:4-Black Hole Quencher 1) and carried out at 23° C.for 15 min, or otherwise indicated periods of time. Compoundconcentration was 8.5 μM, unless indicated otherwise, DMSOconcentrations in wells was 1.7% (v/v). HTS of chemical compoundlibraries was performed in 384 or 1536 well plates using a Perkin ElmerEnvision 2102 multilabel reader. The compounds with an inhibitory effectof 30% or greater were tested further by measuring the concentrationdependence (in a range from 1 nM to 100 μM) of their inhibition ofRAD51. The most potent inhibitory compounds were analyzed further usingnon-fluorescent assays.

D-Loop Assay for RAD51:

The D-loop assay was performed essentially as described previously(Bugreev & Mazin, 2004, Proc. Natl. Acad. Sci. 101:9988-93). To form thenucleoprotein filament, RAD51 protein (1 μM) was incubated with³²P-labeled 90 mer ssDNA (SEQ ID NO:6) (3 μM, nt) in buffer containing25 mM Tris-acetate (pH 7.5), 1 mM ATP, 1 mN CaCl₂, 100 μg ml⁻¹ BSA, 1 mMDTT and 20 mM KCl for 15 min at 37° C. When indicated, chemicalcompounds in question were added in specified concentrations andincubation was continued for 30 min at 37° C. D-loop formation wasinitiated by addition of supercoiled pUC19 dsDNA (50 μM, nt) andcontinued for 15 min. Reactions were stopped by addition of SDS to 1%(w/v) and proteinase K to 880 μg ml⁻¹ followed by incubation for 15 minat 37° C. Samples were mixed with 0.1 vol of loading buffer (70%(v/v)glycerol, 0.1% (w/v) bromophenol blue) and analyzed by electrophoresisin 1% (w/v) agarose-TAE (40 mM Tris-acetate, pH 8.0 and 1 mM EDTA) gels.The yield of joint molecules was expressed as a percentage of the totalplasmid DNA.

To measure the kinetics of D-loop formation, RAD51 protein (0.3 μM) wasincubated with ³²P-labeled ssDNA (SEQ ID NO:6, 90 mer) (0.9 μM, nt) inbuffer containing 25 mM Tris-acetate (pH 7.5), 1 mM ATP, 1 mM CaCl₂, 100μg/ml BSA, 1 mM DTT and 20 mM KCl for 15 min at 37° C. after (FIG. 11A,I) or before (FIG. 11A, II) incubation with Compound B02 (20 μM) forindicated time at 37° C. Then the D-loop formation was initiated byaddition of 15 μM of supercoiled dsDNA (pUC19). The reaction wasdeproteinized and analyzed as described above.

D-Loop Assay for RecA:

To form the nucleoprotein filament RecA protein (1 μM) was incubatedwith 90 mer ³²P-labeled ssDNA (SEQ ID NO:6) (3 μM, nt) in buffercontaining 25 mM Tris-acetate (pH 7.5), 1 mM ATP, 10 mM MgCl₂, 100 μgml⁻¹ BSA, 1 mM DTT, 3 mM phosphoenolpyruvate and 5 U ml⁻¹ pyruvatekinase for 5 min at 37° C. (Mazin et al., 2000, EMBO J. 19:1148-56).When indicated, chemical compounds in specified concentrations wereadded and incubation was continued for 30 min at 37° C. D-loop formationwas initiated by addition of supercoiled pUC19 dsDNA (50 μM, nt) andcarried out for 3 min at 37° C. The DNA products were deproteinized andanalyzed as described above for the Rad51-promoted reaction.

Three Strand Exchange Assay:

RAD51 (1.0 μM) was incubated with indicated concentrations of CompoundB02 in buffer containing 25 mM Tris-acetate (pH 7.5), 250 mM NaCl, 2 mMATP, 1 mM DTT, 10 mM MgCl₂, and 2 mM CaCl₂ for 30 min at 37° C. ThenφX174 circular ssDNA (4.0 μM) was added to the reaction mix to form thenucleoprotein filaments followed by 5 min incubation, then RPA (0.27 μM)was added followed by another 5 min incubation. Reactions were initiatedby addition of ³²P labeled linear φX174 dsDNA (4.0 μM) (DNA form Icleaved by ApaLI endonuclease). The reaction was stopped and DNA wasdeproteinized by addition of SDS to 1% and proteinase K to 880 μg/ml andincubation for 15 min at 37° C. Samples were mixed with 0.1 vol ofloading buffer (70% glycerol, 0.1% bromophenol blue) and analyzed byelectrophoresis in 1% agarose-TAE (40 mM Tris acetate, pH 8.0 and 1 mMEDTA) gels; gels were quantified by using a Storm 840 PhosphorImager(Molecular Dynamics).

DNA Branch Migratin Assay for RAD54:

The partial Holliday junction (PX-junction), in which one of the fourDNA arms is single-stranded (FIG. 6A), was used as a substrate for the4-stranded branch migration promoted by RAD54 (Mazina et al., 2007, J.Biol. Chem. 282:21068-80). PX junctions were constructed in such a way(by incorporating a region of heterology) that allowed branch migrationonly in one direction (Bugreev et al., 2006, Nature 442:590-93). Toproduce PX-junctions, tailed DNA (Oligo 170, SEQ ID NO:9, Oligo 171, SEQID NO:10) (1.45 μM, molecules) was annealed with ³²P-labeled fork DNA(Oligo 71; SEQ ID NO:7; Oligo 169, SEQ ID NO:8) (1.33 μM, molecules) inbranch migration buffer containing 25 mM Tris acetate, pH 7.5, 3 mMmagnesium acetate, 2 mM ATP, 1 mM dithiothreitol, 100 μg ml⁻¹ bovineserum albumin, 10 U ml⁻¹ creatine phosphokinase, 15 mM creatinephosphate for 10 min at 37° C. and then for 10 min at 30° C. Then,branch migration was initiated immediately by addition of the RAD 54(100 nM) to PX-junctions (33 nM, molecules) in branch migrtion bufferand was carried out at 30° C. Aliquots (10 μl) were withdrawn after 0,3, 5, 15, 30, and 60 min. The DNA products were deproteinized bytreatment with stop buffer (1.4% (w/v) SDS, 960 μg ml⁻¹ proteinase K,7.5% (w/v) glycerol, 0.015% (w/v) bromphenol blue) for 5 min at 22° C.and analyzed by electrophoresis in 8% (w/v) polyacrylamide gels (29:1)in 1×TBE buffer (89 mM Tris borate, pH 8.3, and 1 mM EDTA) at 22° C. Thegels were dried on DE81 chromatography paper (Whatman, Kent, UK) andquantified using a Storm 840 PhosphorImager (Molecular Dynamics,Piscataway, N.J.).

Branch Migration Assay:

For RAD51-promoted reaction, RAD51 (1 μM) was incubated with B02 in theindicated concentrations in buffer containing 30 mM Tris HCl, pH 7.5, 10mM MgCl₂, 350 mM NaCl, 2 mM DTT, 2 mM ATP, 8 mM phosphocreatine, and 8units/ml cretine phosphokinase for 30 min at 37° C. Branch migration wasinitiated by addition of ³²P-labeled 3′-joint molecule (0.1 nM,molecule) that were produced by RAD51-promoted reaction and was carriedout for 8 h. For RecA-promoted reaction, RecA (1 μM) was incubated withB02 in the indicated concentrations in buffer containing 25 mM Trisacetate, pH 7.5, 15 mM magnesium acetate, 2 mM DTT, 2 mM ATP, 10 mMphosphocreatine, and 10 units/ml creatine phosphokinase for 30 min at37° C. Branch migration was initiated by addition of ³²P-labeled3′-joint molecules (0.1 nM, molecules) that were produced byRecA-promoted reactin and was carried out for 30 min. The DNA productswere deproteinized and analyzed in 1.5% agarose-TAE gels and quantifiedusing a Storm 840 PhosphorImager (GE Healthcare).

Ethidium Bromide Displacement Assay:

Ethidium bromide (0.5 mg ml⁻¹) was added to pUC19 supercoiled dsDNA (2μg ml⁻¹) in buffer containing 25 mM Tris acetate, pH 7.5, 20 mM NaCl,and 10 μM EDTA followed by 1 min incubation. The fluorescence of thesample was measured using a FlouroMax-3 fluorimeter at an extensionwavelength of 260 nm and an emission wavelength of 546 nm. Then, thesmall molecule inhibitors were added in increasing concentrations andallowed to equilibrate for 1 min, followed by the fluorescencemeasurement.

Calculation of IC₅₀ Value for RAD51 Inhibitors:

IC₅₀ values were calculated using GraphPad Prism V5.0 software and thesigmoidal dose-response function. The data were obtained from threeindependent repeats of experiments.

DNA Binding Assay:

DNA binding assay was performed as described (Bugreev et al., 2005, J.Biol. Chem. 280:26886-95). Briefly, 1.0 μM RAD51 protein and 25 μMCompound B02 were incubated in buffer containing 25 mM Tris-acetate (pH7.5), 2 mM ATP, 100 μg/ml BSA, 1 mM DTT and 2 mM CaCl₂ for 30 min at 37°C., then ³²P-labeled ssDNA (SEQ ID NO:6, 90 mer) (2.5 μM) and NaCl inindicated concentrations were added to the reaction mixture. After 10min, reactions was mixed with 0.2 vol of loading buffer and loaded on10% polyacrylamide gels (135 V, 20 mA) in 0.5×TBE buffer containing 45mM Tris borate (pH 8.3) and 0.25 mM EDTA. Gels were quantified using aStorm 840 PhosphorImager.

ATPase Assay:

RAD51 (1 μM) was incubated with Compound B02 in indicated concentrationsin buffer containing 25 mM Tris-acetate (pH 7.5), 1 mM DTT, 100 μg/mlbovine serum albumin (BSA), 2 mM Mg(OAc)₂, 0.1 mM ATP and 10 μCi [γ-³²P]ATP(6000 Ci/mmole) for 30 min at 37° C. Then ssDNA (#90, SEQ ID NO:6, 90mer) (3 μM, nt) was combined to the reactions. After 1.5 h, the sampleswere applied on polyethyleneimine cellulose strips and subjected to thinlayer chromatography (PEI-TLC) developed in 1 M formic acid/0.5 M LiCl.The fraction of the inorganic [³²P] phosphate (Pi) released from [γ-³²P]ATP was quantified by using a Storm 840 PhosphorImager (MolecularDynamics).

dsDNA Coaggregation Assay:

To measure the effect of Compound B02 on the dsDNA to the secondaryRAD51 binding site, the DNA coaggregation assay was pre-formed asdescribed (Tsang etal., 1985, Biochemistry 24:3226-32). RAD51 protein (1μM) was incubated with ssDNA (#71, SEQ ID NO:7, 94 mer) (3 μM, nt) inbuffer containing 25 mM Tris-HCl, pH 7.5, 1 mM ATP, 100 μg/ml BSA, 1 mMDTT, 20 mM KCl (added with the protein stocks) and 2 mM CaCl₂ for 15 minat 37° C. Then Compound B02 (20 μM) was added to the mixture followed byaddition of NaCl in indicated concentrations and coaggregation wasinitiated immediately by addition of ³²P-labeled pUC19 dsDNA (linearizedby BamHI restriction endonuclease, 25 μM, nt). After 10 min incubationat 37° C. aliquois (10 μl) were withdrawn from the reaction mixture andcoaggregates were collected by centrifugation in 0.5-ml Eppendorf tubesat 15,000×g for 5 min at 21° C. The yield of coaggregates was quantifiedusing a LS 6500 liquid scintillation counter (Beckman). Residualretention of the radioactive DNA on the tube walls, −2-3% of totalradioactivity, was substracted from the measurements.

DR-GFP Assay for Homologous Recombination in Human Cells:

To measure the effect of Compound B02 to the efficiency of DSB- inducedhomologous recombination in human cells a chromosomally located DR-GFPreporter system in 293 human embryonic kidney (HEK) cells was used(Pierce et al., 1999, Genes Dev. 13:2633-38). 293 HEK cells expressingDR-GFP were grown until the log phase. Then cells were harvested andreplated in 6-well plates (TPP, Switzerland) (pre-coated by 0.1% w/vpoly-L-Lysine) with the density of 4.5×10⁵ cells/well; after 24 hr,media were refreshed, then Compound B02 in indicated concentrations wasadded followed by 1 h incubation at 37° C. Then a 200 μl mixturecontaining 2 μg of pCBASce supercoiled plasmid DNA encoding I-SceIrare-cutting restriction endonuclease (Richardson et al., 1998, GenesDev. 12:3831-42) and 6 μl of GenDrill™ transfection reagent (BarnaGenBioScience LLC) in Opti-MEM® I Reduced-Serum (Invitrogen) was added toeach well. After 2 days incubation, cells were trypsinized and dilutedto density of 200 cells/μl in PBS (10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, 137 mMNaCl and 2.7 mM KCl, pH 7.4) for Fluorescence Activated Cell Sorting(FACS) analysis on a Guava EasyCyte Plus System (Millipore, Billerica,Mass).

Effect of Compound B02 on Transfection Efficiency of pMX-GFP Plasmidinto 293 HEK Cells and Expression of GFP Protein:

The log-phase 293 HEK cells containing the DR-GFP reporter wereincubated with Compound B02 in indicated concentrations for 1 h at 37°C. Transfection was carried out by addition of a 200 μl mixturecontaining 2 μg of pMX-GFP supercoiled plasmid DNA encoding GFP proteinand 6 μl of GenDrill™ transfection reagent (BarnaGen BioScience LLC) inOpti-MEM® I Reduced-Serum (Invitrogen) to each well. After 2 daysincubation, cells were trypsinized and diluted to density of 200cells/μl in PBS buffer for Fluorescence Activated Cell Sorting (FACS)anaylsis on a Guava EasyCyte Plus System (Millipore, Billerica, Mass.).

Detectin of Level of RAD51 and SceI Expression by Western Blotting:

HEK-GFP cells were incubated on 10-cm tissue culture plates in DMEM®until ˜70% confluence, then treated with B02 in indicated concentrationsfor 24 h, trypsinized, resuspended in DMEM+, and precipitated bycentrifugation at 1,000×g for 5 min at 4° C. The pellets were washedthree times with cold PBS and then resuspended in lysis buffercontaining 50 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100,50 mM NaF, and 10 mM Na₄P₂O₇, 1 mM PMSF, and proteinase inhibitorcocktail (Roche Applied Science) for 30 min at 4° C. The cell lysateswere centrifuged at 16,000×g, for 15 min at 4° C., and then thesupernatants were collected and protein concentrations were determinedusing the Bradford assay. Aliquots containing 50 μg of total proteinwere analyzed by 12% SDS-PAGE. After electrophoresis, the proteins weretransferred on a polyvinylidene fluoride (PVDF) membrane (Osmonics, Inc)using a Mini Trans-Blot cell (Bio-Rad) at 70 V for 3 h at 4° C., andRAD51 was visualized using anti-RAD51 IgG rabbit polyclonal antibodies(gift of Dr. Etim Golub) (1:5,000 dilution) and horseradishperoxide-conjugated goat antirabbit secondary antibodies (Santa CruzBiotechnology) (1:1000 dilution) in 1×TBST buffer (10 mM Tris-HCl, pH8.0, 150 mM NaCl, 0.1% v/v Tween 20) supplemented with 4% nonfat milk.

To determine the expression level of RAD51 log-phase 293 HEK cellscarrying the DRGFP were incubated on 10 cm tissue culture plates in DMEMmedia supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/mlstreptomycin containing Compound B02 in indicated concentrations cellsreach confluence (˜24 h); to determine the expression level of I-SceIrestriction endonuclease, log-phase cells were treated by Compound B02in indicated concentrations for 1 h, and them transfected with pCBASceplasmid DNA followed by incubation on 10 cm tissue culture plates untilcomplete cell confluence (˜64 h). Then, both pCBASce-transfected anduntransfected cells were trypsinized and resuspended in DMEMsupplemented with 10% FBS, 100 units/ml penicillin and 100 μg/mlstreptomycin. The suspensions were centrifuged at 1000×g for 5 min at 4°C., the pellets were washed 3 times with cold PBS and then resuspendedin lysate buffer containing 50 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM EDTA,1% Triton X-100, 50 mM NaF, and 10 mM Na₄P₂O₇, 1 mM phenylmethylsulfonylfluoride (PMSF) and proteinase inhibitor cocktail (Roche AppliedScience) for 30 min at 4° C. The cell lysates were centrifuged at16000×g, for 15 min at 4° C. The supernatants were collected and proteinconcentrations were determined by the Bradford assay using bovine serumalbumin (BSA) as a standard. Equivalent amounts of proteins were loadedon 12% SDS-PAGE gels and after electrophoretic separation weretransferred on a PVDF membrane (Osmonics, Inc). For lysates fromuntransfected cells, RAD51 protein was probed with anti-RAD51 IgG rabbitpolyelonal antibodies (kind gift of Dr. Efim Golub, Yale University) andhorseradish peroxidase conjugated goat anti-rabbit secondary antibodies(Santa Cruz Biotechnology); for lysates from pCBASec-transfected cells,I-SceI protein containing the HA antigen was probed with HA-Tag (6E2)mouse monoclonal antibodies (Cell Signaling Technology) and horseradishperoxidase-conjugated donkey anti-mouse secondary antibodies (Santa CruzBiotechnology). Immunoblot signals were detected using EnhancedChemiluminescence (ECL) reagent (Pierce). To strip the antibodies, themembranes were incubated for 30 min at 50° C. with occasional agitationin a solution containing 2% SDS, 62.5 mM Tris-HCl, pH 6.8, and 100 mMβ-mercaptoethanol before reprobing with anti-β-actin antibodies.

Clonogenic Survival Assay:

To test the sensitivity of MEF cells to DNA-damaging agents in theabsence or presence of Compound B02 (5 μM), the clonogenic survivalassay was used as described (Essers et al., 1997, Cell 89:195-204).Briefly, MEF cells were trypsinized and the appropriate number of cellswas replated on 10 cm tissue culture dishes. After overnight culture,cells were incubated for 1 h in media containing Compound B02 (5 μM),followed by addition of mitomycin-C (MMC) or cis-dichlorodiamineplatinum (II) (cisDDP) in indicated concentrations and additionalincubation for 1 h. Then the cells were washed 3 times with PBS bufferand incubated in media containing Compound B02 (5 μM) for 7-10 days.Cells were fixed and stained using staining (0.05% crystal violet, 50%methanol in PBS); colonies were counted using a Alphalmager 3400 (AlphaInnotech Inc.). The percent of survival was determined as described(Hall & Giccia, 2005, “Radiobiology for the Radiologist”, 6^(th) Ed.,Philadelphia, Pa.: Lippincott Williams & Wilkins, pp. 30-45). Thepercent of survival obtained from untreated cells was normalized to 100%survival.

Joint Molecule Formation Assay:

For RAD51-promoted reaction, RAD51 (1 μM) was incubated with B02 in theindicated concentrations in buffer containing 25 mM Tris acetate, pH7.5, 2 mM ATP, 275 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, and 100μg/mL BSA for 20 min at 37° C., and the pBSK (+) gapped DNA 4 μM, nt)was added to form nucleoprotein filaments for 15 min. RPA (80 nM) wasadded to the mixture followed by at 10-min incubation. Joint moleculeformation was initiated by addition of 5′-labeled linear pBSK (+) dsDNA(4 μM, nt) (linearized by XhoI) and carried out for 2 h.

For RecA-promoted reaction, RecA (1 μM) was incubated with indicatedconcentrations of B02 in buffer containing 33 mM Tris HCl, pH 7.5, 3 mMMgCl₂, 2 MM DTT, 100 μg/ml BSA, 10 mM phosphocreatine, and 10 units/mlcreatinine phosphokinase for 30 min at 37° C., and then pBSK (+) gappedDNA (5 μM, nt) was added to form the nucleoprotein filaments for 10 min.Joint molecule formation was initiated by addition of 3′-labeled linearpBSK (+) dsDNA (4 μM, nt) (linearized by AlwNI) and single-stranded DNAbinding protein (SSB) (82.5 nM) protein and carried out for 20 min.

In RAD51 and RecA-promoted reactions, joint molecules were deproteinizedby addition of SDS to 1% and proteinase K to 880 μg mL⁻¹ and incubationfor 15 min at 37° C. 0.1 vol of loading buffer (70% glycerol, 0.1%bromophenol blue) was added and joint molecules were either analyzed byelectrophoresis in 1% agarose-TAE (40 mM Tris-acetate, pH 8.0, and 1 mMEDTA) gels and quantified using a Storm 840 PhosphorImager (GEHealthcare); or joint molecules were passed twice through S-400 Spincolumns (GE Healthcare) equilibrated with 25 mM Tris-acetate, pH 7.5, at23° C., and used as substances in branch migration reactions.

Measurement of B02 Binding to RAD51 by SPR:

Experiments were performed using the ProteOn XPR36 SPR array system withProteOn Manager Software version 3.0 (Bio-Rad). ProteOn GLH sensor chipswere preconditioned with two short pulses each (10 s) of 50 mM NaOH, 100mM HCl, and 0.5% SDS. The system was then equilibrated with PBS-T buffer(20 mM Na-phosphate, 150 mM NaCl, and 0.005% Tween 20, pH 7.4).Individual ligand flow channels were activated for 5 min at 25° C. witha mixture of 1-ethyl-3-[3-dimethylaminopropyl carbodiimidehydrochloride) (0.2 M) and sulfo-N-hydroxysuccinimide (0.05 M).Immediately after chip activation, either RAD51 (6.8 μM in 10 mM sodiumacetate, pH 4.5), RecA (2.6 μM in 10 mM sodium acetate, pH 4.5), orHIV-INL4-3 capsid protein (0.5 μM in 10 mM sodium acetate, pH 5.0) wasinjected across ligand flow channels for 5 min at a flow rate of 30 μlmin-1. Excess active ester groups on the sensor surface were capped by a5-min injection of 1 M ethanolamine-HCl (pH 8.5). This resulted in thecoupling of RAD51, RecA, and CA to 14,000, 9,000, and 17,000 RUs(response unit, which is an arbitrary unit that corresponds to 1pg/mm2), respectively. The standard deviation in the immobilizationlevel from the six spots within each channel was less than 4%. B02 inindicated concentrations in buffer S containing 25 mM Tris-acetate, pH7.5, 100 μM CaCl₂, 3% DMSO, and 100 μM ATP (when indicated) was injectedover the control or RAD51 chips at a flow rate of 100 μl min-1, for2-min association phase, followed by a 15-min dissociation phase at 25°C. using the “one-shot” functionality of the ProteOn. Specificregeneration of the surfaces between injections was not needed owing tothe nature of the interaction. Data were analyzed using the ProteOnManager Software version 3.0 (Bio-Rad). The responses of a bufferinjection and responses from the reference flow cell were subtracted toaccount for nonspecific binding. Experimental data were fitted globallyto a simple 1:1 binding model. The average kinetic parameters(association [ka] and dissociation [kd] rates) generated from three datasets were used to define the equilibrium dissociation constant (K_(d)).

Effect of B02 on RAD51 Oligomerization:

RAD51 (50 μg) was incubated in either the presence or absence of B02(200 μM) in 100 μl buffer E (35 mM Tris-acetate; pH 7.5, 150 mM NaCl,10% v/v glycerol 1 mM ATP, 1 mM CaCl₂, and 14.3 mM β-mercaptoethanol)for 10 min at 37° C. The samples were then centrifuged for 15 min at16,000×g, 4  C. Supernatants were loaded on a Superose 6 10/300 GLcolumn (GE Healthcare) equilibrated with buffer E and eluted with a flowrate of 0.2 ml/min at 4° C. The gel filtration molecular weight markerkit (29-669 kDa, Sigma) was used for column calibration. The 0.3-mlfractions were collected and analyzed by electrophoresis in a 12%SDS-PAGE gel (100 V, 90 min).

Transfection of HEK Cells with siRNA:

To suppress RAD51 expression, HEK cells were transfected with 100 nMRAD51 siRNA (sc-36361; Santa Cruz Biotechnology). Briefly, cells (1×10⁶)were seeded in a 3.5-cm tissue culture plate and incubated overnight,then the medium was removed and the cell layer was washed three timeswith PBS. Transfection was carried out by adding 1 ml transfectionsolution containing 100 pmol (1.25 μg) siRNA and 10 μl siRNAtransfection reagent (sc-29528; Santa Cruz Biotechnology) in Opti-MEM IReduced-Serum medium (Invitrogen) to each plate. Six to 7 hours aftertransfection the medium was replaced with fresh DMEM+. Twenty-four, 48,72, and 96 h after transfection, the RAD51 level was determined usingWestern blotting. As a specificity control scrambled siRNA (sc-37007;Santa Cruz Biotechnology) was used instead of RAD51 siRNA.

RAD51 Foci Formation:

2×10⁵ log phase HEK-GFP cells were seeded in 3.5-cm tissue cultureplates on glass coverslips protreated with 0.01% poly-L-lysine (Sigma)and grown overnight. The cells were then incubated with B02 (50 μM) orwithout B02 for 2 h at 37° C. Cells were then exposed to 0.5 Gy IR usinga Primus Linear Accelerator (Siemens) at 6 mV, 3 Gy/min followed by a2-h incubation at 37° C. Cells were washed with phosphate-bufferedsaline (PBS) (8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 138 mM NaCl and 2.7 mMKCl, pH 7.4), extracted with PBS containing 0.2% Triton X-100 and 1 mMphenylmethanesulfonylfluoride (PMSF) for 5 min at 4° C., and fixed withPBS supplemented with 3.0% formaldehyde and 2.0% sucrose for 10 min.Cells were permeabilized with PBS containing 0.5% Triton X-100 for 5 minand blocked with PBS containing 3% BSA and 0.05% Tween 20 for 30 min at23° C. Cells were treated with the polyclonal rabbit RAD51 antibodies(1:500 dilution) (a gift from Dr. Golub, Yale University) in PBScontaining 3% BSA and 0.05% Triton X-100 overnight at 4° C., and thenwith Alexa Fluor 488 donkey antirabbit IgG (H+L) antibodies (1:1000dilution) (Invitrogen) for 1 h at 23° C. followed by a 5-min stainingwith 500 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) in PBS containing0.05% Tween 20. Samples were washed in PBS containing 0.05% Tween 20,mounted in VectaShield™ (Vector Laboratories), and sealed with nailpolish. The fluorescence images were obtained using an Olympus IX70inverted microscope and iVision-Mac™ software (BioVision).

Example 1

Fluorescence-Based DNA Strand Exchange Assay

A FRET-based DNA strand exchange assay suitable for HTS of largelibraries of chemical compounds was developed. In this assay, RAD51promotes DNA strand exchange between homologous synthetic ssDNA anddsDNA substrates. The dsDNA carries fluorescein (FLU), a fluorescentdonor group, and black hole quencher 1 (BHQ1), a non-fluorescentacceptor group, which were attached to the 5′- and 3′-ends of thecomplementary ssDNA strands, respectively (FIG. 1A). In this dsDNAsubstrate, the fluorescence at he FLU group is quenched by BHQ1 throughFRET. As a result of RAD51-promoted DNA strand exchange, theFLU-carrying DNA strand is displaced from the dsDNA that carries theBHQ1 and the fluorescence of the FLU group increases (Parkhurst &Parkhurst, 1995, Biochem. 34:293-300; Parkhurst et al., 2001, Biopol.61:180-200).

Using this assay the kinetics of RAD51-promoted DNA strand exchange wasmeasured. RAD51 was loaded on the homologous ssDNA (SEQ ID NO:2; 48-mer)(denoted as “Homologous DNA”) to form the nucleoprotein filament. Then,fluorescently labeled dsDNA (SEQ ID NO:4-Black Hole Quencher 1; andfluorescein-SEQ ID NO:3) was added to the filament to initiate DNAstrand exchange. After a 1 h incubation the fluorescence intensity at521 nm increased approximately 20-fold (FIG. 1B). To ensure that theobserved fluorescence increase resulted from DNA strand exchange, acontrol was run in which the RAD51 filament was assembled onheterologous ssDNA (SEQ ID NO:5, 48-mer) (denoted as “HeterologousDNA”). Since DNA strand exchange does not occur between heterologous DNAmolecules (Shibata et al., 1979, Proc. Natl. Acad. Sci. 76:1638-42), noincrease in fluorescence was expected. Indeed, in the case ofheterologous DNA the intensity of fluorescence remained almost constantduring the 1 h of incubation (FIG. 1B). Thus, the results validated theFRET-based assay to measure the DNA strand exchange activity of RAD51.

Example 2

HTS of the NIH Small Molecule Repository

The NIH Small Molecule Repository (202,556 compounds) was screened forRAD51 inhibitors using the FRET-based assay described above. 174positive hits that showed more than 30% inhibition of the DNA strandexchange activity of RAD51 were detected (hit rate, 0.09%) using aPerkin Elmer Envision 2102 multilabel reader. Measuring theconcentration dependence of RAD51 inhibition by these compounds allowedthe identification of the seventeen most potent inhibitors as candidatesthat warranted further analysis (FIG. 2).

Example 3

Analysis of RAD51 Inhibitors Using the D-loop Assay

To validate the hits identified in the primary FRET-based assay, theseventeen selected compounds (FIG. 2) were further analyzed using theD-loop assay. In the D-loop assay, DNA strand exchange was promoted byRAD51 between homologous ³²P-labeled ssDNA and pUC19 supercoiled plasmidDNA (FIG. 3A). The products of this reaction, joint molecules, alsoknown as D-loops (called after the displaced DNA strand that is formedin joint molecules during DNA strand exchange), were identified byelectrophoresis in a 1% agarose gel. First, the inhibitory effect ofeach of the seventeen compounds on the efficiency of D-loop formationwas measured. Eleven of the seventeen compounds inhibited D-loopformation by more than 50% (FIGS. 3B and 3C, Table 1). Thus, 65% of thecompounds identified in the primary assay were validated by thesecondary assay. In addition, using a fluorescence intercalator(ethidium bromide) displacement assay, Compounds A05, A06, A14, and A15were found to be DNA binders (data not shown). These compounds alongwith Compounds A08, A11, and B01, which showed relatively modestinhibition, were not analyzed further. Then, the IC₅₀ values for thefour most potent remaining inhibitors of the RAD51 DNA strand exchangeactivity were determined using the D-loop assay. The IC₅₀ for CompoundsA03, A04, A10, and B02 were 33.2 μM, 5.0 μM, 26.6 μM, and 27.4 μM,respectively (FIG. 4; Table 2).

TABLE 1 Effect of selected compounds (100 μM) on the D-loop formationpromoted by RAD51 Compounds Joint Molecules (%) DNA binding^(a) Control30.3 ± 1.9 A03  5.4 ± 1.9 A04  1.4 ± 0.1 A05  1.6 ± 0.2 + A06  3.5 ±0.1 + A07 29.6 ± 0.1 A08 10.0 ± 0.8 A09 32.9 ± 3.5 A10  6.7 ± 2.5 A1110.2 ± 1.7 A12 19.2 ± 3.4 A13 27.8 ± 3.8 + A14  3.8 ± 0.1 + A15  4.2 ±0.7 + B01 11.0 ± 2.3 B02  6.0 ± 0.5 B04 24.8 ± 0.1 B05 20.1 ± 0.8^(a)DNA binding was determined by the fluorescent interculator (ethidiumbromide) displacement assay.

TABLE 2 IC₅₀ values for selected RAD51 inhibitors IC₅₀(RAD51) IC₅₀(RecA)IC₅₀(RecA)/ Compound (μM) (μM) IC₅₀(RAD51) A03 33.2 187.3 5.6 A04 5.05.7 1.1 A10 26.6 35.3 1.3 B02 27.4 >250 n/a C3a 15.3 >100 n/a C3b27.3 >100 n/a

Example 4

Analysis of a RAD51 Inhibitor Using the Homologous Pairing and ThreeStrand Exchange Assay

The effect of Compound B02 on homologous pairing activity of RAD51 byD-loop assay was analyzed. RAD51 can promote homologous searching andpairing of ssDNA on supercoiled plasmid dsDNA, which contains homologoussequences of ssDNA (FIG. 3A). In this assay, ³²P-labeled ssDNA (SEQ IDNO:6, 90 mer) (0.9 μM, nt) and supercoiled dsDNA (pUC19, 15 μM) wasemployed as DNA substrates. Compound B02 was found to efficientlyinhibit the strand exchange promoted by RAD51 with aconcentration-dependent manner (FIG. 3D, lane 1-9). And alsointerestingly, the Compound B02 did not inhibit D-loop formation of RecAprotein, the prokaryotic homolog of RAD51 (FIG. 3D, lane 10-18). IC₅₀for RAD51 is 10.4 μM; while for RecA protein is more than 100 μM (FIG.3E), which indicates that Compound B02 is a very specific inhibitor forRAD51.

A strong inhibition of Compound B02 on RAD51 was also observed in threestrand exchange assay, in which a strand exchange between circular φX174ssDNA and homologous linearized φX174 dsDNA (linearized by ApaL1endonuclease) was promoted by RAD51 (FIG. 10A). Yields of jointmolecules (JM) and nicked circular DNA (NC) were compared among thedifferent doses of treatment with Compound B02 (FIG. 10B). The resultsshow that formation of both joint molecules and nicked circular DNAdecreased with an increasing of Compound B02 concentration. IC₅₀ for thewhole product (JM+NC) was 26.03 μM, which was comparable with the valueobserved in D-Loop assay if 1 μM RAD51 was used (data was not shown).IC₅₀ for the JM was 23.3 μM and for NC was 26.7 μM, suggesting that theCompound B02 has the same inhibition to JM and NC formation.

Example 5

Specificity of RAD51 Inhibitors

RAD51 shares structural and functional similarity with RecA from E.coli; both proteins promote DNA strand exchange in vitro and share 30%homology. Using the D-loop assay, the effect of the selected RAD51inhibitors on the DNA strand exchange activity of RecA was evaluated.Compound A03 showed some moderate specificity for RAD51 (FIG. 5A), withthe IC₅₀ 5,6-fold lower for RAD51 than for RecA (Table 2). Compounds A04and A10 inhibited RAD51 and RecA with a neatly equal efficiency (FIGS.5B & 5C; Table 2). Finally, Compound B02 showed the highest specificityfor RAD51 (FIG. 5D); the IC₅₀ for RAD51 was 27.4 μM, whereas for RecA nosignificant inhibition of DNA strand exchange was observed up to 250 μMof Compound B02 (Table 2).

The inhibitory effects of Compound A03, A04, and A10 compounds wereevaluated to determine whether they are specific for the proteins of theRad51/RecA family or have a broader specificity. To address thisquestion the effect of the inhibitors on human RAD54, a Swi2 protein,which does not share structural homology with the proteins of theRecA/RAD51 family, were tested (Mazin et al., 2010, DNA Repair (Arnst)9:286-302). RAD54 promotes branch migration of Holliday junctions, aprocess in which one DNA strand is progressively exchanged for another(FIG. 6A). The effect of Compounds A03, A04, and A10 compounds on theRAD54 branch migration activity was tested using a ³²P-labeledoligonucleotide-based cruciform DNA substrate, known as the partialHolliday junction or PX-junction (FIG. 6A). Of the four compoundstested, Compounds A03, A10 and B02 were shown not to significantlyinhibit RAD54 in the range of concentrations from 0 to 200 μM (FIG. 6B).However, Compound A04 did have an inhibitory effect on the RAD54 branchmigration activity (IC₅₀=2.6 μM) (FIG. 6C).

Thus among tested compounds, Compound B02 was identified as a specificinhibitor of human RAD51. Compounds A03 and A10 inhibited bothRAD51/RecA family proteins, RAD51 and RecA. Compound A04 showed thebroadest inhibitory spectrum by inhibiting all three tested proteins:RAD51, RecA and RAD54.

Example 6

Blockage of the RAD51-ssDNA Filament Formation at the Presynaptic Stage

To explore the mechanism how Compound B02 inhibits the homologous pairand strand exchange, three experiments were carried out.

Firstly, the time course of D-loop formation was compared with differentorder of addition of Compound B02 and ssDNA (FIG. 11A). In Protocol (I),20 μM Compound B02 was added after the filament formation; and then thesamples were incubated for indicated times before the initiation ofD-loop formation by addition of dsDNA. In Protocol (II), RAD51 was firstincubated with 20 μM Compound B02 for indicated time; and then ssDNA wasadded and incubated for 15 min at 37° C. to form the filament before theinitiation of D-loop formation by addition of dsDNA. The results showthat, in both Protocols (I) and (II), the efficiency of D-loop formationdecreased in the presence of Compound B02 (FIG. 11B, lane 1-7 and 8-14),while the efficiency of D-loop formation increased or keep stable in theabsence of Compound B02 (data not shown). Furthermore, the relativeinhibition (FIG. 11C), which was expressed as the ratio of the jointmolecules with Compound B02 treatment to those without Compound B02treatment, shown that in the presence of Compound B02 the efficiency ofD-loop formation in Protocol II (FIG. 11B, lane 8-14) reduced muchfaster than that in Protocol I (FIG. 11B, lane 1-7).

The results suggest the Compound B02 can either block the assembly ofthe filaments if added before filament formation or disrupt theassembled filaments if added after the filament formation. However, alower portion of filaments was disrupted if Compound B02 was added afterthe filament formation, owing to the high stability of nucleoproteinfilaments.

Secondly, the effect of Compound B02 on assembly of RAD51-ssDNAfilaments was tested using ssDNA binding assay. In this assay, RAD51 (1μM) was incubated with 25 μM Compound B02 , which was comparable withIC₅₀ obtained from D-loop assay if 1 μM RAD51 was used (data was notshown). Then ssDNA (SEQ ID NO:6, 90 mer) (3 μM, nt) was added in thepresence of indicated concentration of NaCl. The binding of ssDNA ofRAD51 protein was impaired with the challenge of increasingconcentration of NaCl. If Compound B02 could disrupt the RAD51-ssDNAfilaments, more ³²P-labeled free ssDNA dissociating from the filamentswould be observed. The results (FIG. 12) show that without Compound B02,the ratio of ssDNA in complex decreased from 100% (0 mM NaCl) to 71%(200 mM NaCl), only 29% loss of filaments; while with Compounds B02, theratio of ssDNA decreased from 80% (0 mM NaCl) down to almost 0% (200 mMNaCl), which means all the filaments were disrupted in the presence of200 mM NaCl. Besides, even without NaCl challenge, there was still 20%loss of filaments in the presence of Compound B02. These results suggestthat Compound B02 can disrupt the RAD51-ssDNA filaments.

Thirdly, the effect of Compound B02 on the ATPase activity of RAD51 wastested by concentration titration of Compound B02. As illustrated inFIG. 13, Compound B02 can inhibit the ATPase activity of RAD51 with aconcentration-dependent manner, and IC₅₀ is 37.6 μM. The DNA-dependentATPase activity of RAD51 is correlative with the binding of DNA on theprotein. Therefore, the inhibition of Compound B02 on ATPase activity ofRAD51 also suggests that Compound B02 can disrupt the RAD51-ssDNAfilaments.

Example 7

Disruption of the Binding of dsDNA on RAD51-ssDNA Filament

In the RAD51 protein, there are two specific DNA-binding sites: theprimary DNA binding site and the secondary binding site (Sung et al.,2003, J. Biol. Chem. 278:42729-32). In the presynaptic stage of strandexchange, the RAD51-ssDNA filament was assembled by binding of ssDNAinto primary binding site of RAD51. The search of DNA homology was doneby reiterative binding and release of duplex DNA on and from thesecondary binding site of RAD51 in RAD51-ssDNA filaments until thehomology was located. So the secondary binding site is alsoindispensable for the strand exchange. DNA coaggregation assay was usedto test the effect of Compound B02 on binding of duplex DNA on thesecondary hinding site of RAD51.

In this assay, the RAD51-ssDNA filament was assembled by incubation of 1μM RAD51 with 3 μM ssDNA (SEQ ID NO:7, 94 mer). RAD51 primary bindingsite was saturated by ssDNA at this DNA concentration. Then the filamentwas mixed with 25 μM dsDNA (pUC19, linearized by BamHI) and challengedby NaCl in the absence or presence of 20 μM Compound B02. If CompoundB02 can disrupt the binding of dsDNA on secondary binding site of RAD51,the DNA coaggreations will be more unstable in Compound B02 presencesamples than in Compound B02 absence samples, and the percent of dsDNAin coaggregations (FIG. 14) will be lower in Compound B02 presencesamples than in Compound B02 absence samples. As expected, much lowerpercent dsDNA in coaggregations was observed in Compound B02 presencesamples. Even without the challenge of NaCl, the ratio of dsDNAcoaggregations in Compound B02 presence samples was only 8%, which is ⅕of that in Compound B02 absence samples. These results suggest thatCompound B02 can disrupt not only the binding of ssDNA on primarybinding site of RAD51 but also the binding of dsDNA on secondary site ofRAD51, both of which are critical to the homologous recombination.

Example 8

Inhibition of HDR of a Chromosomal DSB

DR-GFP assay can be used to monitor HDR of chromosomal DSB by fusion aDR-GFP report gene construct in chromosome of cells. In DR-GFP construct(FIG. 15A), SceGFP gene, which encodes green fluorescent protein (GFP),was disabled by insertion of an 18-bp recognition site for I-SceIcleavage into SceGFP gene. A DSB can be generated by expressing of thetransfected pCBASec plasmid, which encodes I-SceI endonuclease. Repairof the I-SceI induced DSB by recruitment of IGFP (an internal GFP genefragment truncated at both ends) as a template (FIG. 8A) gives rise to afunctional GFP gene. This HDR event can be scored by green fluorescencein individual cells, using flow cytometric analysis. Consequently theeffect of Compound B02 on the HDR of a chromosome in cells can beclearly addressed. The pCBASec plasmid was transfected in DR-GFP 293cells in the presence of 0 μM, 5 μM, 10 μM and 20 μM of Compound B02. Tovalidate the gene transfection, the cells transfected with pMX-GFP,which encodes functional GFP protein, were used as a positive control.And notransfected parental cells were used as a negative control.Compound B02 can reduce the yield of GFP positive cells by aconcentration-dependent manner. As shown in FIG. 15B, the yield of GFPpositive cells decreased from 3.28% in non-treatment cells to 0.40% inthe cells treated with 20 μM of Compound B02, an 8-fold reduction wasobserved, suggesting that as a RAD51 inhibitor, Compound B02 canefficiently disrupt the HDR promoted by RAD51. But this claim can beeasily invalidated by two possibilities: (1) Compound B02 inhibits thetransfection or expression of pCBASce, which just produces less DSB thanthat in the absence of Compound B02, and consequently less DSB repairwill produce lower level of GFP positive cells; (2) Compound B02inhibits the expression RAD51 in the cells. Recently published paper hasreported a Histone deacetylase (HDAC) inhibitor, which indirectlyinhibited HDR by reducing the expression of RAD51 (Adimaolan et al.,2007, Proc. Natl. Acad. Sci. USA). To exclude these possibilities, firstthe effect of Compound B02 on the plasmid transfection was tested. ThepMX-GFP plasmid, which encodes the functional GFP protein, was used as areporter. After transfection of pMX-GFP plasmid into DR-GFP 293 cells,the expression of functional GFP protein was monitored by flowcytometric analysis. The cells transfected with pMX-GFP plasmid, butwithout treatment of Compound B02 were used as a control. It wasobserved that Compound B02 has no inhibition to transfection incomparison to control cells. The ratio of GFP positive cells iscomparable between the cells in control group (31.52%) and the cellswith 20 μM Compound B02 (31.47%) (FIG. 15C). Furthermore, the expressionof I-SceI protein in DR-GFP 293 cells was assessed with or withouttreatment of Compound B02 using western blot assay. The results showthat Compound B02 did not affect the expression of I-SceI protein (FIGS.16A & 16B). Next, a western blot assay was carried out to test theeffect of Compound B02 on the expression of RAD51, the data show thatthe expression of RAD51 was comparable between the cells withouttreatment of Compound B02 and cells treated with 20 μM Compound B02(FIGS. 16C and 16D). The results of the two experiments above suggestthat the reduction of yield of GFP positive cells is attributed to theinhibition of Compound B02 on RAD51 protein, which is a key protein inthe HDR of Chromosomal DSB.

Example 9

Sensitivity of MEF Cells to Double-Strand Break (DSB) Induced Agents

Based on the observation that Compound B02 can inhibit the homologousrecombination in vitro, it is quite interesting to explore if CompoundB02 has inhibition on homologous recombination in cell level. Herein thesensitivity of MEF cell lines (wide type and Tp53−/−) to the DNAcross-linking agents cisDDP and MMC in the absence and presence ofCompound B02 was analyzed by using colongenic survival assay. Beforethis experiment, the toxicity of Compound B02 to the cell lines selectedwas first assessed. No decrease of cellular viability with the treatmentup to 5 μM Compound B02 was observed. Therefore this concentration wasselected for the colongenic survival assay. As shown in FIG. 17, in thepresence of 5 μM Compound B02, both cell lines were more sensitive tothe cisDDP and MMC, which suggest that Compound B02 may reduce theresistance of cells to DNA damage agents by inhibiting the DSB repair.Some SSB-induced agents such as MMS induce DNA damage. Failure to repairthe SSB could produce stalled replication fork, and collapsing of thestalled replication fork could finally cause DSB. PARP-1 protein is akey protein in BER pathway, which is very important in SSB repair. Hereto validate inhibition of Compound B02 to RAD51 protein in mammaliancells, the sensitivity of MEF cells to DNA damage agents MMS was tested.MEF cells were treated by MMS to induce the SSBs in the presence of 5 μMCompound B02 or 1 μM of a PARP-1 inhibitor, AZD2281 (olaparib or4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fluorophenyl]methyl(2H)phthalazin-1-one)or both. The cells treated only with MMS were used as a control. Theresults show that in the presence of 1 μM AZD2281, the cells are moresensitive to MMS than that in the absence of AZD2281 (FIG. 18). Thesensitivity enhancement may be attributed to that the treatment ofAZD2280 inhibits the BER, which blocks the repair of the SSB induced byMMS. Additionally, if in the presence of both Compound B02 and AZD2282,the cells were more sensitive to MMS than that only in the presence ofAZD2281, suggesting that not only the SSB repair was blocked, meanwhilethe repair of DSB induced by the collapsing the stalled fork was alsoblocked, the data shows evidence that the Compound B02 inhibits therepair of DSB in the cells by inhibition of the RAD51 activity. Also theenhancement of sensitivity was observed in the cells with co-treatmentof the MMS and Compound B02. This could happen because even in thenon-treated cells there is certain amount of stalled fork which couldcollapse and induce DSH. The enhancement of sensitivity may response tothe inhibition of Compound B02 to DSB repair.

Example 10

SAR Analysis of Compound B02

For SAR analysis of Compound B02, a 16-compound library of B02derivatives was selected (FIG. 7A). According to the structuralfeatures, the 16 compounds were sorted in 5 groups. In group 1 (compoundC1): (E)-2-(pyridin-3-yl) vinyl was removed with (E)-2-(R₁-substitutinggroup) vinyl; in group 3 (compounds C3a to C3e): benzyl was replaced by6-iodo-quinazolin-4(3H)-one; in group 5 (compound C5): the core,quinazolin-4(3H)-one was replaced by 6-iodo-quinazolin-4(3H)-one, aswell as (E)-2-(pyridin-3-yl) vinyl was replaced by (E)-2-(pyridin-2-yl)vinyl.

The inhibitory effect of these B02 derivatives (50 μM) on the DNA strandexchange activity of RAD51 was determined using the D-loop assay. Theonly position of BO2 that could tolerate modifications and still inhibitRAD51 was the benzyl (compound group 3) (FIG. 7B and 7C). Moreover, onlycompound C3a (R₂ ethyl) and compound C3b (R₂=m-methyl phenyl) retain theinhibitory effect, the substitutions of other groups (C3d, R₂=phenyl andC3e, R₂=methyl) or at the different positions (C3c, R₂=p-methyl phenyl)eliminates the inhibition, suggesting that the inhibition is tightlyrelated to the size and the steric conformation of the substitutinggroup. All other tested replacements in the groups 1, 2, 4, and 5eliminated RAD51 inhibition. The high sensitivity of the RAD51inhibition to B02 modifications suggests specific interactions betweenthe inhibitor molecule and RAD51 protein.

Example 11

Inhibitor Optimization

From the library of B02 derivatives, two compounds (C3a and C3b) thatinhibited RAD51 were identified (FIG. 7). For these two inhibitors, theIC₅₀ values of the RAD51 DNA strand exchange activity and theirselectivity for RAD51 were determined using the D-loop assay (FIG. 8).The IC₅₀ for C3a and C3b are 15.3 μM and 27.3 μM, respectively (FIG. 8B7 8C). To evaluate the selectivity of the inhibitors the effect of C3aand C3b on the DNA strand exchange activity of RecA were determined. Theresults show that compounds C3a and C3b in concentrations up to 100 μMdo not inhibit RecA (FIG. 8; Table 2).

Thus, the current results demonstrate the feasibility and efficiency ofthe HTS approach for discovery of novel selective inhibitors of RAD51, akey protein of homologous recombination and the repair of DNA doublestrand breaks and interstrand crosslinks. Further experiments mayinclude establishing the mechanism of specific RAD51 inhibition byselected small molecule compounds (B02, A03, A10) and examining theeffect of these compounds on the RAD51-dependent homologousrecombinations and DNA repair in human cells.

Example 12

Binding to RAD51 and Inhibition of Its Activities

RAD51 and its E. coli homologous RecA possess DNA strand exchange andDNA branch migration activities. The effect of B02 (FIG. 19A) on theseactivities of both proteins was examined. pBSK (+) gapped and lineardsDNA substrates that allow separate analysis of DNA strand exchange andbranch migration promoted by RAD51/RecA were used (FIG. 19B). At thefirst step, RAD51/RecA promoted DNA strand exchange between gapped DNAand linear DNA substrates resulting in formation of joint molecules.Joint molecules were then purified and used as substrates for RAD51/RecAbranch migration. In accord with previous data showing specificinhibition of RAD51 by B02 in the D-loop assay, B02 (10-100 μM)inhibited DNA strand exchange promoted by RAD51 (FIGS. 19C-19D), but notby RecA (FIGS. 19D & 20A). B02 (10-100 μM) was found to inhibit the DNAbranch migration activity of RAD51 (FIGS. 19E-19F). The inhibition wasspecific, as B02 did not inhibit the branch migration activity of RecA(FIG. 19F & 20B). The IC₅₀ value of RAD51 inhibition by B02 was 35 μMfor both DNA strand exchange and DNA branch migration. Next, it wastested whether RAD51 inhibition is caused by the direct interaction ofB02 with RAD51 . Using the surface plasmon resonance (SPR) technique,B02 (6.25-50 μM) was shown to bind to RAD51, but not to RecA (FIGS.21-22). For B02 binding to RAD51 in the absence of ATP, kinetic valueswere as follows: k_(a)=4.5 (±0.3)×10³ M⁻¹ s⁻¹; k_(d)=2.5 (±0.3)=10⁻²s⁻⁴; K_(d)=5.6 μM. Using the ethidium bromide displacement assay B02 wasshown not to bind DNA. Thus, B02 inhibited DNA strand exchange andbranch migration activities through direct and specific binding toRAD51.

Example 13

Disruption of the RAD51 Foci Formation

B02 was tested to determine whether it can inhibit RAD51 activities inthe cell. In response to DNA damage, RAD51 accumulates in distinctnuclear structures, known as foci. Because RAD51 foci colocalize withssDNA formed in the cell after DNA damage, it is thought that the focirepresent RAD51 complexes with DNA repair intermediates. B02 was foundto inhibit RAD51 foci formation induced in 293 human embryonic kidney(HEK) cells by IR. In the presence of B02 (50 μM), the fraction of cellswith RAD51 foci (≧1 focus) was decreased in non-irradiated cells(15±13%) (FIGS. 23A-23B); the average number of RAD51 foci per nucleusdecreased 4.4-fold, from 53±11 to 12±4 (FIG. 23C). At lowerconcentrations (20 μM), B02 also inhibited IR-induced RAD51 fociformation, however the inhibitory effect was smaller.

Example 14

Increase in Cell Sensitivity to DNA-Damaging Agents

B02 was examined to determine whether it can enhance cell sensitivity toDSB- and ICL-inducing anticancer agents, cisplatin and MMC. Using theclonogenic survival assay it was found that in the presence of B02 (5μM) mouse embryonic fibroblasts (MEF) became approximately 17- andfivefold more sensitive to cisplatin (32 μM) and MMC (1 μM),respectively (FIGS. 24A-24B). Because, p53 protein is commonly mutatedin many human cancers, the effect of B02 on Tp53^(−/−) MEF was alsotested. It was found that the sensitivity of Tp53^(−/−) MEF to cisplatinand MMC increased in the presence of B02 similarly to wild type cells(FIG. 25). In these experiments, 5 μM B02 was used, a concentration atwhich B02 alone did not have a substantial effect on the viability ofwild type or Tp53^(−/−) MEF (FIG. 24C). Inhibitory effect of B02 on cellsurvival observed in co-treatment experiments could be due to depletionof RAD51 that translocated to the sites of DNA damage. This hypothesiswas tested using HEK cells in which the RAD51 expression level wasdecreased by siRNA (FIG. 26). The results showed that indeed thecombination of specific RAD51 siRNA and B02 more strongly sensitized HEKcells to cisplatin than did each of these reagents alone (FIG. 24D). Theminimum incubation time with B02 that was required for cells'sensitization for cisplatin was determined. These data indicate that10-12 h of incubation was required to increase the sensitivity of HEKcells for cisplatin (FIG. 24E). After 16 h, the maximal sensitivity wasreached. Thus, B02 causes cell sensitization to cisplatin and MMC,indicating the ability of B02 to inhibit RAD51-dependent DSB repair inthe cell.

As described herein, the mechanism of RAD51 inhibition by B02 and itsability to inhibit RAD51 homologous recombination and DNA repair incells was analyzed. B02 was found to bind to RAD51 and inhibit its DNAstrand exchange and branch migration activities with high specificity,as it does not affect E. coli RecA, a demonstrated that B02 can inhibitRAD51-dependent HR events in the cell and promote cell killing bycytotoxic DSB and ICL-inducing agents.

Because the RAD51-ssDNA filament plays a critical role in HR, itsformation is tightly regulated by various factors that either enhance orinhibit RAD51 binding to ssDNA. These data demonstrated that B02 alsoinhibits RAD51 filament formation. These results showed that B02 impairsRAD51 filament formation by targeting protein-DNA interactions. Thefilament formation involves binding ssDNA to the RAD51 primary site.Using a coaggregation assay, it was found that B02 also inhibits dsDNAbinding to the secondary RAD51 site, which normally occurs during thesearch for homology.

To address of whether B02 inhibits RAD51-depending HR and DNA repair inthe cell, several cell-based assays were carried out. First, it wasfound that B02 inhibited formation of RAD51 foci in response to IR,which is thought to reflect RAD51 accumulation at the sites of damagedDNA and formation of RAD51-DNA complexes during recombinational DNArepair. Then, using a chromosomally integrated GFP reporter, it wasshown that B02 decreased, up to eightfold, the frequency of DSB-inducedHR in human cells. It was also demonstrated that B02 increased cellsensitivity to ICL- and DSB-inducing agents, cisplatin and MMC. Finally,it was found that a combination of B02 with PARP1 inhibitor AZD2281increased cell sensitivity to an alkylating agent (MMS) to a greaterextent than does AZD2281 alone. The finding that PARP1 inhibitorsenhance the effect of B02 on cell sensitivity to DNA damage isconsistent with inhibition of HR by B02. In the co-treatmentexperiments, B02 showed activity at lower concentrations (5 μM) than inother biological assays, likely due to the depletion of RAD51 thataccumulates at the sites of DNA damage. Indeed, it was found thatdepletion of RAD51 with siRNA had an additive effect with B02 treatment.Moreover, RAD51 is not a canonical enzymes; the DNA strand exchangeassay requires rather high RAD51 concentrations (stoichiometric relativeto DNA substrates). Concerning RAD51 foci formation, it is worth nothingthat each RAD51 focus involves thousands of RAD51 monomers, therefore adecrease in their number in each focus may not have been readilydetectable by immunostaining and required higher B02 concentrations(20-50 μM). Overall, these results demonstrated that B02 inhibitsRAD51-dependent HR and DSB repair in mammalian cells (FIG. 27).

Because small-molecule inhibitors may be applied in a cell cycle and ina concentration dependent manner, they are especially useful foranalysis of proteins essential for cell viability, like RAD51. Byapplying B02 for different periods of time after DNA damage by cisplain,a maximal time for which DNA repair can be delayed before the cellsstart dying was determined.

These results indicated that a combination of inhibitors that targetalternative DNA repair pathways, e.g., RAD51-dependent andPARP1-dependent DNA repair, can be especially efficient for sensitizingcancer cells for radio- and chemotherapeutic agents. Targeting RAD51 mayrepresent an important strategy to specifically eradicate cancer cells.Consistent with the compensatory role that HR may play in cancer cells,RAD51 was found to be overexpressed in many tumors.

These results demonstrate that B02 inhibitor of RAD51 can efficientlysuppress DSB dependent HR in the cell. The inhibitor can be used for theanalysis of RAD51 cellular functions and for development of novelanticancer therapies.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of Formula (1):

wherein: R¹ is pyridinyl; R² is selected from the group consisting ofC₁-C₆ alkyl, —(C₁-C₆)alkylene-phenyl, and —(C₁-C₆)alkylene-phenylwherein the phenyl is substituted with a C₁-C₆ alkyl; R³ is H or a saltthereof.
 2. The pharmaceutical composition of claim 1, wherein R2 ismethyl, ethyl, n-propyl, isopropyl, benzyl, benzyl substituted on thephenyl ring with a C1-C6 alkyl.
 3. A pharmaceutical compositioncomprising a compound selected from the group consisting of:(E)-3-benzyl-2-(2-(pyridine-3-yl)vinyl)quinazolin-4(3H)-one(1a)

(E)-3-ethyl-2-(2-(pyrindin-3-yl)vinyl)quinazolin-4(3H)-one(1b)

and salts thereof and any mixtures thereof.
 4. The pharmaceuticalcomposition of claim 1, further comprising a chemotherapeutic agent. 5.The pharmaceutical composition of claim 4, wherein the agent is selectedfrom the group consisting of an alkylating agent, antimetabolite,anthracycline, plant alkaloid, plant terpenoid, topoisomerase inhibitor,antineoplastic agent, and any combinations thereof.
 6. Thepharmaceutical composition of claim 3, further comprising achemotherapeutic agent.
 7. The pharmaceutical composition of claim 6,wherein the agent is selected from the group consisting of an alkylatingagent, antimetabolite, anthracycline, plant alkaloid, plant terpenoid,topoisomerase inhibitor, antineoplastic agent, and any combinationsthereof.